Chem 431 Lab Manual W 2016

CHEM 431 LAB MANUAL
TABLE OF CONTENTS
Safety Rules............
Lab Equipment.......
The Format of Formal Lab Reports ...
Automatic Pipettes: Instruction for Use .
Lab #1: Checking the Accuracy and Precision of Pipettes ...
10
Lab #2: Quantitative Measurement of Proteins ....
14
Lab #3: Electrophoresis of Serum Proteins Using SDS-PAGE ....
25
Drying Polyacrylamide Gels in Cellophane.
33
Analyzing the gel using the Image J Software ..
35
Lab #4: Restriction Enzyme Analysis of Circular DNA ...
43
Lab #5: Isolation of an Enzyme: Acid Phosphatase
51
Lab #6: Characterization of Acid Phosphatase ....
65
Instructions for the oral presentation ..
67
Appendix I: Introduction to UV-Vis Spectrophotometers ..
68
Instructions for UV-visible Spectrophotometers (Beckman DU-520) ..
70
Appendix II: Linear Regression Analysis: Excel 2007
71
Appendix III: Analyzing Agarose Gel Electrophoresis of DNA .....
74
SAFETY
C.W.U. Chemistry Department
Teaching Laboratory Safety Rules
The Chemistry Department is committed to providing a safe environment for students and staff. However,
laboratory safety is a mutual responsibility and requires full participation and cooperation of all involved students, TAs and instructors. The following lab safety rules have been established for your protection as a
student in the Chemistry Department. These rules will be rigidly and impartially enforced. Noncompliance
may result in a grading penalty and/or dismissal from lab. A statement of agreement to compliance with
the lab rules is included on the lab check-in sheet and must be signed prior to beginning lab.
Personal Protection
Safety Goggles must be worn at all times in active chemistry labs. This is the policy of Central Washington
University and also a state requirement. The goggles must be of the indirect vented type and must meet ANSI
Z87.1 specifications. The bookstore has approved goggles safety glasses are not acceptable.
Do not remove the plastic vent covers from the goggles.
The safety of wearing contact lenses in chemical laboratories has been hotly debated over the last several years.
Both the ACS and OSHA have issued statements indicating that contact lenses can be worn IF AND ONLY IF
proper protective eyewear is also worn. The Chemistry Department recognizes that some eye conditions require
contacts for certain vision correction therapies. However, students who choose to wear contacts must recognize
the inherent risks they are difficult to remove if chemicals get in the eye, they have a tendency to prevent
natural eye fluids from removing contaminants, and sudden displacement can cause visual problems that create
additional hazards. Soft contact lenses are especially problematic because they can discolor and also absorb
chemical vapors causing damage before the wearer is alerted to the problem. If you choose to wear contacts,
please tell your lab instructor and write Contact Lenses Wearer by your signature on your lab check-in form.
Appropriate gloves will be provided when needed. Use of gloves is required for handling certain chemicals. Gloves
are very expensive. Do not change gloves needlessly. Do not wear gloves outside the lab where they can
contaminate doors and other surfaces contacted. Wash hands after removing gloves, before leaving the lab.
Appropriate clothing is required. Your clothing is a barrier between your skin and chemicals. All skin from
shoulders across the collar bone to toes must be completely covered (excluding hands). Lab coats are required
during the teaching labs by everyone at all times. They can be purchased at the student book store or
online. WAC 296-800-160 Code of Federal Regulations, Title 29 CFR, Part 1910, Subpart 1
Shoes must be worn and must completely cover all parts of the feet. WAC 296-800-160 Code of Federal
Regulations, Title 29 CFR, Part 1910, Subpart 1
Long hair must be tied back.
Do not eat, drink (including sport bottles), or store food in the labs.
Smoking or use of other tobacco products is prohibited.
Wash hands after working with chemicals.
Never mouth pipette use a pipette bulb.
Do not taste chemicals. Check odors only if instructed by your Instructor- by wafting gently toward your nose
with your hand.
It is the recommendation of this department that all students of reproductive age, especially women who have
recently conceived or are anticipating conception during the quarter, discuss the course content and reagents with
their physician if concerned about reproductive toxins.
For your safety, if you have a medical condition that results in seizures, blackouts, etc. (e.g., from epilepsy,
diabetes) please inform your instructor who will direct you to the department safety representative. This
information will be kept confidential. If you wish to seek accommodations due to a disability or to address
functional limitations, please contact Disability Services at 509-963-2214.
General Lab Rules
No horseplay or running. Keep aisles clear - push stools against the bench when not in use. Store backpacks in
cubicles provided.
No music allowed in student labs. Radios (including IPods) and other entertainment devices are not permitted.
Read all instructions carefully and plan your work ahead of time. Understand the experiment and if in doubt, ask.
Unsupervised laboratory work is not permitted. Do not perform unauthorized experiments.
No personal phone calls are allowed on lab phones - public phones are available on the first floor, east side.
Cell phones should be turned off while in lab.
Updated 8/12/2015
(over)
Chemistry lab computers are for chemistry business only - no Internet surfing or checking email.
Handle chemicals carefully. Note the colored labels affixed to the bottles. The Chemistry Department follows the
J.T. Baker Saf-T-Data tm labeling system.
Colors represent hazard classes as follows:
Orange
Minimal hazard, general precautions
Blue
Toxic
Yellow
Reactive, unstable
Yellow Stripe
Incompatible with other Yellows
Red
Flammable
Red Stripe
Incompatible with other Reds
White
Corrosive, usually acids
White Stripe
Incompatible with other Whites, usually bases
Treat chemicals with respect and understand the chemicals you are using. Material Safety Data Sheets (MSDSs)
are available at the MSDS Workstation outside Room 311 and online. Do not remove the MSDSs from the binders;
bring the binders to the Chem office (Room 302) to request a copy.
Dispose of waste chemicals in the appropriate labeled waste containers never dispose of chemicals down the
drain unless directed to do so by the lab instructor.
Leave the lab area clean. Put equipment and chemicals away, wipe off the bench top and clean the balance pans.
JUST IN CASE
In case of an emergency, contact your TA and instructor immediately and be prepared as follows:
Spills Assess the situation and notify the instructor. Small low level hazard spills can be cleaned up with the
help of the TA using the spill kit in the prep room. If the spill is flammable, shut gas off immediately. If any
chemical is spilled on the skin or splashed in the eyes, remove all jewelry and contaminated clothing and flush the
affected area with water for 15 min.
Glass Breakage DO NOT HANDLE broken glass with bare hands, consult TA or instructor on how to clean-up.
Put the broken glass in the Broken Glass Container not in the trashcan. Fill out a breakage slip and place it the
box in the prep room. The TA will use the mercury spill kit to clean up thermometers never put them in the
trashcan.
Personal Injury all injuries and near misses must be reported.
Minor injuries may be treated as follow:
Cuts Rinse with water. Band-Aids are available from your TA.
Thermal Burns Flush with cold water. Do not cover, Report to your instructor/ TA immediately.
Chemical Burns Flush for 15 min. using sink, shower, or eyewash, Report to your instructor/ TA immediately.
Fill out a student accident report (forms located in First Aid drawer) and return signed form to the Chemistry
Stockroom (across from SCI 315).
Power Outage Await instructions. If power is not restored in 15 min., your TA will help you to begin
shutting down the lab. Put all chemicals away. Turn off gas and electrical equipment. Pull hood sashes down.
Fire Alarm Sounds Indicates imminent danger. Close chemical containers, shut off gas and electricity, exit
from labs down the stairwell do not use elevators. Your instructor will provide specific information concerning the
remainder of the lab and re-entry into the building. Faculty, staff and TAs will assemble on the lawn on the North
side of the Japanese Garden. DO NOT CLEAN UP OR PUT THINGS AWAY EVACUATE IMMEDIATELY!
If you have any questions, the following people are your safety resources:
- your instructor
- the Chemistry Instructor
- the Chemistry Stockroom Manager Tony Brown SCI 303, 963-1303
- the Chemistry Department Safety Representative Ian Seiler SCI 315, 509-607-1620
- the campus Industrial Hygienist James Hudson 963-2338
- the campus Environmental Health & Safety Officer Ron Munson 963-2252.
For additional department safety information visit the department safety web page at
http://www.cwu.edu/chemistry/laboratory-safety
Updated 8/12/2015
Lab Equipment
Beakers
Graduated cylinder
Polypropylene funnel
Microspatula
Spin bar
Wash bottles
Erlenmeyer flask
Three-prong clamp
Filter flask
Safety bulbs
Scoop spatula
Rubber policeman
Clamp holder
Buchner funnel
Micropipette
Mohr pipet
Thermometer
Test tube
Pipette tip holder
Test tube brush
Glass rod
Neoprene adapters
Test tube clamp
Test tube rack
Watch glass
Microfuge tube rack
5
The Format of Formal Lab Reports
Two experiments will be described in a formal report organized in a research publication format.
During the laboratory period data and observations must be recorded in a carbonless copy
laboratory notebook (purchased at the University Bookstore, e.g., Saunders Publishing). Write
simply and concisely in the passive or third person voice. In your report be sure to include
answers to questions and any information or calculations specified in the lab book for each
experiment. Always identify any unknown by number or letter. While you will work with a
partner at times, each lab report must be written up individually. Each lab report must contain
the following six parts presented in the order listed below:
1. Title of the experiment and Name of the author (lab partners names in parentheses).
2. Introduction: Clearly state the purpose of the experiment. State the question to be
answered or investigated and any background needed to introduce the subject. State
the chemical principles involved in the techniques being used.
3. Methods and Materials: If you followed directions exactly, this may be stated by
referencing the handout sheets. However, if changes were made in any materials or
procedures you must specify these in detail.
4. Results: Data must be presented in tables or figures that are numbered and titled. The
tables and figures should be formatted in a manner similar to what is found in the peerreviewed literature. Provide a sample calculation if a series of the same calculation is
used, but list the series of calculated results in the table or figure. All numerical data
must have units. Tables require column titles with units. Figures must have axes
labeled with units. Absorbance values and wavelengths at which the absorbance was
measured must be clearly stated on figures. A brief statement or summary of the results
observed must also be included in this section. Where multiple determinations are
made, means and standard deviations of the mean must be reported.
5. Discussion and Conclusions: State and discuss the significance of your observations.
Were your observations surprising? Explain any deviations from expected results. State
your confidence in your conclusions based on the precision and accuracy of your data
and possible sources of error.
6. References and Appendix: Cite three peer-reviewed papers from the literature that is
relevant to the lab. Yellow pages of your laboratory notebook must be turned in with
your report showing the observations and calculations from the laboratory.
Automatic Pipettes: Instructions for Use

You have three micropipettes that you will use for dispensing precise volumes of liquids.
It is crucial that you learn how to adjust and use these pipettes properly, as your results
will depend upon it. These pipettes are air displacement pipettes and work by creating a
vacuum that draws up a specified volume of liquid. Gaining proficiency with pipettes is
one of the most important skills for biochemists and your success in this course will
depend on your ability to pipette accurately. For more information on how this type of
pipettes work, you can follow this link. The three pipettes that you have in your drawer
dispense volumes within discrete ranges:
100 1000 L
called the p1000 pipette
20 200 L
2 20 L
Reminder: 1 mL = 1000 L
Pipettes tend to be more accurate towards the top of their ranges. Therefore, although
you can use either the p200 or the p20 pipette to dispense 20 L, the p20 pipette will
likely be much more accurate at this volume.
PARTS OF THE MICROPIPETTES
Before beginning to use the pipettes, it is
best to familiarize yourself with the basic
parts (shown in the figure on the right).
The operation button is used to aspirate
(draw up) liquid into disposable tips that
fit over the ends of the barrels and then
to dispense the liquid. The tip eject button
is to get rid of the used pipette tip. The
calibration knob is used to adjust the
volume of liquid that the pipette is set to
dispense. The volume to which the
pipette is set is viewed in the volume setpoint window. The following page
describes how to read this window. Note:
you can distinguish between the pipettes
in three ways, by the labeling on the
operating button, by the color of the
operating button, and by the size of the
barrel.
Reading the volume set-point window

People that are new to using micropipettes
often find reading the volume set-point
window to be confusing, partly because
the windows look a little different for the
various pipettes. The volume set-point
windows for the three different size
pipettes are shown to the right. The red
lines on the pipettes have been
accentuated here - note there is no
horizontal red line on the p200 pipette.
p20 pipette
The top two digits tell the number of
microliters that the pipette will dispense,
while the number below the red line
indicates the tenths of microliters. The tick
marks between the tenths indicate hundredths of microliters. The example window
indicates that the pipette is set to a volume of 19.50 microliters (you can read two
decimal places with the p20 pipette).
p200 pipette
The three digits on the p200 pipette indicates the number of microliters. The tick marks
between the bottom digits indicate tenths of microliters. The example window indicates
that the pipette is set to a volume of 98.0 microliters (you can read one decimal place
with the p200 pipette).
p1000 pipette
The number above the red line indicates the milliliters that the pipette is set to. This digit
will read either zero or one, as the maximum volume that it can dispense is one milliliter.
The two digits below the red line indicate the hundreds and tens of microliters being
dispensed. The tick marks between the tens indicates the individual microliters being
dispensed. This pipette is set to dispense 650 microliters (you cannot read any digits to
the right of the decimal point with the p1000 pipette).
METHOD FOR USE

1) Use the information above to select the correct size pipette. Turn the calibration
knob until the display reads the correct volume. NEVER USE A PIPETTE OUTSIDE
THE RANGE FOR WHICH IT IS SPECIFIED TO BE USE.
2) Press the barrel of the pipette down onto the correct size of disposable tip. The p20
and p200 can use the same size tip (although not all tips fit both size pipettes), while
the p1000 always uses a larger tip. Make sure that the tip forms a complete seal
around the barrel of the pipette. NEVER USE A PIPETTE WITHOUT A TIP
ATTACHED as serious damage will result to the pipette and the incorrect volume of
liquid will be dispensed. If you accidentally pipette liquid into the barrel of the pipette,
immediately inform your instructor so that they can clean and/or repair the pipette.
3) Depress the button on the top of the pipette to the first stop. Then, holding the
pipette vertically, immerse the tip into the liquid to a depth of 3-5 mm. It is important
that you do not submerge more of the tip into the liquid, as liquid will stick to the
outside of the tip, be dispensed into the target vessel, adversely affecting your
pipetting accuracy.
4) Slowly release the button to aspirate the specified volume of fluid into the tip. It is
crucial that you do this fairly slowly (especially with the larger pipettes) as releasing
the button too quickly can cause air bubbles to form and may result in liquid being
pulled into the barrel of the pipette.
5) Pull the pipette straight up to pull the tip from the liquid. If there is any liquid on the
outside of the tip, you may gently touch the tip to the inside of the container from
which you are pipetting in order to remove the excess liquid. Do not wipe the outside
of the tip with a tissue, Kim wipe, or paper towel.
6) Place the tip into the vessel that you wish to dispense the liquid into. If there is
already liquid in the vessel, then immerse the tip into the liquid to a depth of 3-5 mm
and then press the button all the way to the second stop. Remove the tip while
holding the bottom down to the second stop. If there is not already liquid in the
vessel, then gently press the tip against the inside of the bottom of the vessel and
press the button to the second stop and remove the pipette while holding down the
button.
7) Press the eject button to eject the tip into an appropriate container.
ADDITIONAL OPERATING HINTS

A) Make sure that the tip is in place and correctly fitted. A poor fitting tip will leak and
will result in inaccurate pipetting.
B) Aspirating and dispensing of liquids should be done in a slow, controlled manner.
Pipetting too quickly results in inaccuracy and can potentially cause liquids to be
sucked up into the pipette and damage it.
C) Always hold the pipette vertically when in use. Do not hold the pipette horizontally or
set the pipette down on the bench when it is holding liquids, as the liquids can run
into the barrel of the pipette, causing contamination/damage. This is especially
important for the p1000 pipette.
D) If you are pipetting viscous liquids, such as buffers containing glycerol, it may be
helpful to pre-wet the inside of the tip by pipetting up and down once with the liquid
before performing the actual volume measurement.
E) It is often necessary to mix the components as you add them together. The two most
commonly used techniques for mixing while adding components include pipetting up
and down and stirring with the pipette tip. Remember: if you are working with
enzymes or other properly folded proteins, you will want to mix very gently.
10
Checking the Accuracy and Precision of Pipettes

Water has a density that varies with temperature, with room temperature water (22C)
having a density of 0.99777 g/mL. You will use this constant to determine the accuracy
of the pipettes that you have been assigned. The protocol that you will use will also give
you a measure of your pipetting precision. Remember: accuracy is how close the
average volume the pipette dispenses is to an intended volume whereas precision is
how close the volumes that the pipette dispenses are to each other. You may have
seen the following example describing accuracy and precision:
Equipment and Reagents Needed
A clean 50 mL beaker containing 40 mL of distilled water (white tap)

A screw top vial (obtained from the instructor)
The p1000, p200, and p20 micropipettes
Disposable tips for the three pipettes
A 100-200 mL beaker that will be used to collect used pipette tips
Analytical balance
Calculator
Microsoft Excel can make the calculations much easier to complete.
11
Protocol
Step 1:
Collect the equipment and reagents listed and find an analytical balance to
work at.
Step 2:
Pour or pipette about a milliliter of water into the screw top vial. Place the vial
on the analytical balance and tare (zero) the balance.
Step 3:
Check the accuracy of each pipette at two different volumes (according to the
table below) to be sure that it is calibrated and functioning properly. When
dispensing the water into the vial, make sure to place the tip into the water in
the vial and then press the operating button to the second stop. Remove the
tip from the water while holding the button down to the second stop.
Step 4:
Enter your data into Microsoft Excel and calculate the mean mass of the
water pipette, the standard deviation (SD) of the mean, the coefficient of
variation (CV), and the accuracy of each pipette for the two volumes used.
The CV should be less than 10%. Consult with your instructor if the CV is
greater than 10%.
CV calculation: SD / mean X 100%
Accuracy calculation:
1) Calculate theoretical mass of water by multiplying the volume (in mL) by
0.99777 g/mL (for example, 0.950 mL X 0.99777 g/mL = 0.948 g)
2) use the following equation to calculate the accuracy:
100% [ABS(mean mass - theoretical mass) / theoretical mass X 100%]
(ABS means take the absolute value of the value in parentheses)
An example Excel spreadsheet is posted to the course blackboard

site. Use it at your own risk. Whenever you use an excel
spreadsheet to calculate your data, double check that the formulas
are correct and that your values are being calculated correctly. Also,
double check that you have entered your data correctly.
12
Data Collection Sheet

p1000 pipette
volume = 950 L
mass (g) ________
________
________
________
________
________
________
________
________
________
Mean = _________
SD =
CV =
Accuracy = ___________
_________
___________
volume = 525 L
mass (g) ________
________
________
________
________
________
________
________
________
________
Mean = _________
SD =
CV =
Accuracy = ___________
_________
___________
p200 pipette
volume = 200 L
mass (g) ________
________
________
________
________
________
________
________
________
________
Mean = _________
SD =
CV =
Accuracy = ___________
_________
___________
volume = 105 L
mass (g) ________
________
________
________
________
________
________
________
________
________
Mean = _________
SD =
CV =
Accuracy = ___________
_________
___________
13
p20 pipette
volume = 19.5 L
mass (g) ________
________
________
________
________
________
________
________
________
________
Mean = _________
SD =
CV =
Accuracy = ___________
_________
___________
volume = 7.5 L
mass (g) ________
________
________
________
________
________
________
________
________
________
Mean = _________
SD =
CV =
Accuracy = ___________
_________
___________
14
Quantitative Measurement of Proteins

Spectrophotometry and colorimetry are analytical methods of measuring the
amount of light absorbed by a substance in solution. These methods are commonly
used to measure the quantity of biochemical materials. All substances in solution
absorb light of certain wavelengths and then transmit the light at different wavelengths.
Absorbance is a physical property of a substance just like the melting point or boiling
point. Absorbance can be used to measure the amount of a given substance in solution
by utilizing the Beer-Lambert law:
A = lC
where
A
l
C
=
=
=
=
absorbance at the given wavelength

molar extinction coefficient
path length in cm (almost always 1)
molar concentration of chromophore
The Beer-Lambert law requires the use of a specified wavelength of light. Generally, the
wavelength of light used is the wavelength at which the substance has the greatest
absorbance (the peak) is used and this can be determined by performing a scan over a
given range of wavelengths (shown in Figure 1).
Figure 1. An absorption spectrum of a given substance over a

range of wavelengths from 195 nm to 315 nm. The maximum
absorbance for this substance is approximately 234 nm. If the
concentration of this substance is known, the resulting
absorbance at this wavelength can be used to calculate the
molar extinction coefficient (). Scan taken from V.A. McKie, et.
al (2001) Biochem. J. 355: 167-177.
If the extinction coefficient for a substance at the maximum absorbance is known
and the path length is fixed, the concentration of the substance can be determined. The
extinction coefficient may be obtained from the literature or determined by measuring
the absorbance at different concentrations of the substance. A plot of the absorbance
versus concentration should give a linear plot whose slope is the molar extinction
coefficient (for that unit of protein concentration) when the cell length is 1.00 cm.
Absorption occurs when photon energies in the visible or ultraviolet (UV) regions
cause electronic transitions in a molecule. The absorption of visible and ultraviolet light
by organic compounds usually occurs when there is unsaturation in the molecule. A
15
specific group of atoms having unsaturation and absorbing light is called a chromophore
or chromophoric group. Common chromophoric groups include carbon-carbon double
and triple bonds, carbonyl, carboxyl, amide, azo, nitrile, nitroso, nitro, imidazole, indole,
purine, and pyrimidine groups. Any molecule containing one or more such groups has
an absorption band somewhere in the visible or ultraviolet regions. Conjugated double
bonds also contribute to specific absorption bands, causing a decrease in the energy
required for a transition and producing a peak at higher wavelengths.
Chemical and enzymatic reactions are sometimes designed to generate a
substance that has an absorption maximum in the visible or UV range; such substances
can readily be quantified, providing a convenient way to measure the reaction rate. As
you will see later this quarter, this may involve the addition of compound that indicates
oxidation/reduction, dehydration/condensation, or the addition of a complexing or
chelating agent. The type of reaction is determined by the particular chemistry of the
substance being measured.
In the following set of exercises, you will use three different protein assay
techniques to quantitate the concentration of proteins in samples of unknown
concentrations. It is important that you understand that these three techniques
quantitate the total amount of protein in the samples and are not able to determine the
concentration of a specific protein from a mixture of polypeptides. Other techniques,
with ELISA (enzyme-linked immunosorbant assay) being the most common, are used to
quantify the content of a specific protein in a mixture of proteins. The majority of these
more precise techniques utilize the specific binding of antibodies in order to distinguish
between the given protein and the other nonspecific proteins present in the sample.
16
Bradford Method to Measure Protein Concentration

The laboratory practices of protein purification, enzyme assays, or other biochemical
and cellular analyses require a rapid and sensitive method for the measurement of
protein concentration. The Bradford Method1 for protein quantitation has gained wide
acceptance in the biochemical literature. It is based on the binding of a dye to protein
with a change in dye color upon protein binding. Coomassie Brilliant Blue G-250 (Fig.
1.) is red in solution and blue when complexed with a protein. Coomassie brilliant blue
binds to positively charged side groups on amino acid residues in the protein, especially
to arginine, and to aromatic amino acids. The binding of the dye to protein is a rapid
process that takes 10 to 15 minutes. The protein-dye complex then remains stable in
solution for a sufficient amount of time to complete the assay, approximately 60 min.
Due to the high extinction coefficient of the protein-dye complex, it is a sensitive assay
allowing measurement of protein at the g / mL level. You can read more about this
protein assay by consulting the Bio-Rad website. Bio-Rad is a company that sells the
Coomassie Brilliant Blue G-250 (Figure 2).
Figure 2. Coomassie Brilliant Blue G-250
Reference
1. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:
248-54.
17

A beaker that will be used to as a waste container
1.6 mL microcentrifuge tubes
Microcentrifuge rack
Bio-Rad Bradford Reagent
0.01% w/v (0.1 mg/mL) Bovine Serum Albumin (BSA)
Sample of unknown BSA concentration
Approximately 20 mL of distilled H2O (from white taps) in a clean beaker
Vortexer
Timer
Plastic cuvette
UV-Vis spectrophotometer
Microsoft Excel
Protocol
Note: Refer to the Appendix for instructions for use of the UV-Vis
spectrophotometer.
Step 1:
Prepare BSA samples for making a standard curve by obtaining 1.0 mL of the
0.01% BSA stock solution and making the following dilutions in 1.6 mL
microcentrifuge tubes according to the table below. Before you begin
pipetting, label the caps of your microcentrifuge tubes with: 0, 2, 4, 6,
8, 10, 12. Pipette 1.0 mL of distilled water and dispense it into the tube
labeled 0. This tube will be used as your blank. For the remaining tubes
add the water first, followed by pipetting the BSA directly into the water
(pipette up and down several times to get all of the BSA out of the pipette tip).
Mix the components by inverting the tubes 10 times.
H2O
mL
1.000
0.980
0.960
0.940
0.920
0.900
0.880
Step 2:
Stock BSA volume

mL
0.000
0.020
0.040
0.060
0.080
0.100
0.120
[BSA] after dilution

g protein /mL
0.00
2.00
4.00
6.00
8.00
10.0
12.0
Label the top of three fresh microcentrifuge tubes with: U1, U2, and U3.
Obtain your unknown sample from the refrigerator (It will be labeled as Br#;
with # representing a number). Make sure you write down the number of your
unknown. Without changing the tip, pipette 1.0 mL of your unknown sample
into the three tubes labeled U1, U2, and U3. You will assay the
18
concentration of the same unknown three times (a triplicate determination).
Step 3:
Transfer by pouring 3.0 mL of Bradford reagent (it is located in the refrigerator

and has a yellow label that says Bio-Rad Protein Assay Reagent) into 10 mL
graduated cylinder. You may then transfer the 3 mL of Bradford reagent into a
small clean beaker for pipette access. Use the p1000 pipette to add 0.225 mL
of the Bradford reagent to all the tubes in Steps 1 and 2, i.e., the water blank,
the known BSA standards, and the unknown BSA samples to bring the total
volume up to 1.225 mL. Mix by vortexing or inverting 10 times. Important: the
Bradford method is time-sensitive and the color reaction changes over time.
Thus you must work in a timely manner.
Step 4:
Incubate the Bradford reagent and protein mixtures for 10 minutes on the
bench top (at room temperature). This is a good time to review the amino
acids. Be sure to measure the absorbance of your solutions in an efficient
manner within 15 minutes after the incubation period.
Step 5:
Take your samples, a water bottle, a waste container, and a disposable

cuvette to a spectrophotometer. Check that the spectrophotometer is set to
read 595 nm. Pour the contents of the tube labeled 0 into the cuvette and
place the cuvette in the spectrophotometer. Make sure that the cuvette is
positioned so that the clear windows are on the left and right side, not to the
front and back.
Step 6:
Close the door and zero (blank) the spectrophotometer. The instrument
should now read 0.000. Once the instrument is zeroed, read the absorbance
at 595 nm of the standard curve protein samples from lowest to highest
concentration, then read your unknowns. Use one cuvette for all of your
measurements, rinsing with water in between samples (make sure to give the
cuvette a good shake to remove water from the cuvette or tap it on a set of
paper towels). Pour the next solution to be measured into the cuvette, place it
into the spectrophotometer, and record the absorbance in your laboratory
notebook. As you work please wipe up any spills with a paper towel.
Step 7:
Plot absorbance at 595 nm (y-axis) versus protein concentration in g/mL (xaxis) of the samples of known protein concentration. Use Excel linear
regression analysis to determine the best straight line to fit the data points,
the equation of the line and the R-squared value. Using the equation of the
line and the unknown sample absorbance value, calculate the unknown
protein concentration for each sample, and then the average and standard
deviation and coefficient of variation. Be sure to identify by number the
unknown sample you used.
19
Ultraviolet Absorption to Measure Protein Concentration

Proteins have an absorption spectrum in the ultraviolet region. Below 230 nm,
the absorption of a protein rises rapidly, reaching a maximum around 190 nm due to the
absorption of the peptide bond. Other substances, such as carboxylic acids, buffer ions,
and alcohols, also absorb in this region and interfere with the measurement, making it
less specific for proteins.
Proteins also have an absorption peak near 275-280 nm due to their aromatic
tyrosine and tryptophan residues; the molar extinction coefficients of tryptophan and
tyrosine at 280 nm are 5502 and 1209, respectively (phenylalanine has a molar
extinction coefficient of only 2 at 280 nm). Each protein has its own extinction
coefficient, which varies with its amount of tyrosine and tryptophan. The absorbance
measurements at 280 nm for 1% (w/v) solutions of protein vary from 5 to 60, with many
proteins having values around 10. Absorbance measurements at 280 nm are frequently
used to obtain an elution profile of a chromatographic separation of proteins on a
column. The method is relatively fast, sensitive, easily automated, and (most
importantly) nondestructive. A disadvantage of the method is that many other
compounds also absorb in the 280 nm region, especially nucleic acids, which have a
maximum absorbance around 260 nm. Pure proteins typically have a 280 nm / 260 nm
absorbance ratio of about 1.6, whereas pure nucleic acids have a ratio of about 0.5. An
initial test of the purity of a protein solution may be obtained by measuring and
calculating the 280/260 ratio. You will calculate the 280/260 ratio of a solution of DNA
and protein to determine their relative purity.

1% w/v (10 mg/mL) Bovine Serum Albumin (BSA)
0.25% BSA
0.01% DNA solution
Unknown BSA sample (for UV!)
Squirt bottle filled with dH2O
quartz cuvette
Microsoft Excel
Warning: quartz cuvettes are very fragile and expensive (~$300 each).
Rinse the cuvette out with mild soapy water (and then dH2O) prior to and
after use. Do not use a paper towel to dry the cuvette.
20
Protocol
spectrophotometer.
Part 1:
Step 1:
Gather the following reagents and equipment: a squirt bottle containing dH2O,
a test tube holding ~3-5 mL of 0.25% BSA, a test tube holding ~3-5 mL of
0.01% DNA solution, a quartz cuvette, and a beaker to hold waste material.
Take these materials to an available UV-Vis spectrophotometer.
Step 2:
Use the squirt bottle to fill the quartz cuvette with ~2 mL of dH2O and place
the cuvette in the chamber of the UV-Vis spectrophotometer. Perform a
baseline (blank) scan from 240-320 nm. This will take about 30 seconds.
Step 3:
Pour the water into the waste beaker and then pour ~2 mL of the 0.25% BSA
solution into the cuvette. Press the Scan button. Note the large absorbance
band or peak at 280 nm.
Step 4:
Use the cursor keys to move the vertical line across the screen to the 280 nm
and 260 nm positions. Write down the absorbance shown on the screen for
both wavelengths. You will use these values to calculate a 280/260 ratio for
protein.
Step 5:
Pour out the BSA solution into the waste beaker and rinse out the cuvette
twice with dH2O. Pour ~2 mL of the 0.01% DNA solution into the cuvette and
repeat Steps 3 and 4. You will use these values to calculate a 280/260 ratio
for DNA.
Step 6:
Repeat step 5 with your unknown solution so that you can calculate its
280/260 ratio.
Use this data to calculate the ratio of the absorbance at 280 nm to that at 260 nm
for each solution to determine relative purity.
Part 2:
Step 1:
Obtain 2.0 mL of 1.00% (w/v) BSA (10 mg/mL) from the laboratory
refrigerator. Label 1.6 mL microcentrifuge tubes with the concentrations listed
in the following step.
Step 2:
Use the 1% BSA solution to prepare 1.5 mL of each of the following dilutions:
0.05%, 0.10%, 0.15%, 0.20%, and 0.25% BSA. Use the dilution equation:
CiVi=CfVf, where Ci = initial concentration and C f = final concentration and Vi
and Vf are initial and final volumes, respectively.
Step 3:
Take the following items to an available UV-Vis spectrophotometer: your

diluted BSA samples, your unknown sample, a squirt bottle with dH2O, a
21
quartz cuvette, a waste beaker for rinsing the cuvette and your p1000
pipette with a tip. Set up the spectrophotometer (See appendix) so that it is
reading a single wavelength of 280 nm.
Step 4:
Use the water from the squirt bottle to blank the spectrophotometer. Then,
pipette your dilutions from the 1.6 mL microcentrifuge tubes into the cuvette
(start with the least concentrated). Read and write down the absorbance for
each sample. Finally, rinse the cuvette once or twice with dH2O and pour ~2-3
mL of your unknown BSA sample into the quartz cuvette and measure and
record its absorbance. Pour out the BSA sample and measure and record the
absorbance for the unknown sample two more times. From these three
values you will calculate the average and standard deviation. Make sure you
write down the number of your unknown sample.
Step 5:
Plot the absorbance at 280 nm versus concentration for the samples of

known concentration. Use linear regression to determine the best straight line
through the data points. From the equation for the line, calculate the
concentration (in %) of the unknown solution.
22
Biuret Method to Measure Protein Concentration

Polypeptides and proteins with two or more peptide bonds give a characteristic
purple color when treated with dilute, alkaline copper sulfate solution. The color is
caused by the formation of a complex of copper (II) ion with four nitrogen atoms, two
from each of two peptide chains (Figure 1). The color intensity is proportional to the
concentration of protein. Ammonia or ammonium ions will interfere with this
determination by complexing the copper of the Biuret reagent and giving a false
positive. The Biuret reaction requires relatively large amounts of protein, 1-20 mg.
Figure 1. Protein-copper complex.

1% w/v (10 mg/mL) Bovine Serum Albumin (BSA)
Biuret reagent
Unknown BSA sample (for Biuret!)
Squirt bottle filled with dH2O
plastic cuvette
Microsoft Excel
23
Protocol
spectrophotometer.
Step 1:
Obtain about 3 mL of 1.00% (10 mg/mL) BSA from the laboratory refrigerator
by pouring into a clean 10 mL graduated cylinder. Label 1.6 mL
microcentrifuge tubes with the concentrations listed in step 2.
Step 2:
Dilute the 1% solution of BSA to prepare 0.600 mL (= 600 L) samples

containing 1.0 mg/mL, 1.5 mg/mL, 2.0 mg/mL, 3.0 mg/mL, 4.0 mg/mL, 6.0
mg/mL, and 8.0 mg/mL of BSA. Prepare these in the 1.6 mL microcentrifuge
tubes.
Step 3:
Pipette 600 L of dH2O into a microcentrifuge tube for a blank and 600 L of
the unknown BSA sample to each of the three tubes labeled as unknown.
Step 4:
Add 900 L of the Biuret reagent to each of the tubes you prepared in Steps 2
and 3, followed by mixing by inversion three times or by vortexing. Incubate
the samples containing Biuret reagent in a 37 C water bath for 10 minutes.
Then place the samples in a room temperature water bath for 5 minutes to
cool or allow them to cool on the bench top.
Step 5:
Take your BSA samples (reacted with Biuret reagent), squirt bottle with dH2O,
p1000 pipette, plastic cuvette and a waste beaker to an open UV-Vis
spectrophotometer. Set the spectrophotometer so that it will read absorbance
at 540 nm.
Step 6:
Use the transfer the contents from the microcentrifuge tube labeled blank to
the cuvette, place it in the spectrophotometer and blank the instrument.
Transfer the contents of the cuvette back to the tube (or to the waste beaker),
then begin reading the absorbance of the standard curve samples. Write
down the absorbance values obtained. Rinse the cuvette out with water and
then read the absorbance of the unknown samples.
Step 7:
Plot the absorbance at 540 nm versus concentration for the samples of

known concentration. Use linear regression to determine the best straight line
through the data points. From the equation for the line, calculate the
concentration (in %) of the unknown solution.
24
Grading Rubric: Quantitative Measurement of Protein (Formal Report)
Introduction (10 points):
State the purpose of this experiment, including a reasonable rationale for why you learned
three different techniques for determining protein concentration (2).
Describe the fundamental chemical principles involved for each of the three protein
concentration assays (2 points each).
Explain the relationship between absorbance and concentration and how this relationship is
used to quantitate an unknown protein sample. Invoke Beers law (2).
Methods (2 points):
Cite the lab manual and note any changes you made.
Results (24 points): Make sure to report the numbers of your three unknowns!
Biuret Assay (7 points)
Table listing test tube contents (protein concentration) and absorbance values (1)
Figure showing absorbance vs. concentration using Excel (1). Your figures should be
labeled with a number (e.g., Fig. 1.) and title just as those found in the peer-reviewed
literature. Give your figure a title that describes the data in the figure or the overall purpose
of the figure.
r2 value of 0.98 or higher (1)
correct calculation of unknown protein conc. from standard curve (1)
accuracy of calculated protein concentration (3 points)
error: <3% for 3 pts; <6% for 2 pts; <10% for 1 pt; <15% for 0.5 pt.
Bradford Assay (7 points)
r2 value of 0.98 or higher (1)
Other points distributed the same way as for the Biuret assay except for accuracy:
error: <9% for 3 pts; <18% for 2 pts; <30% for 1 pt; <45% for 0.5 pt.
UV Assay (10 points)
Part 1 correct calculation of 280/260 ratios for protein and DNA solutions (3)
Part 2 points distributed the same way as for the Biuret assay (7)
Discussion (10 points):
Discuss the important sources of error (2)
Comment on accuracy and precision of results and your resulting confidence in these
results (2)
Compare the 280/260 ratios of the pure DNA and protein samples and of your unknown with
those in the lab manual and comment on your conclusions from the comparison (2).
Calculate the absorbance of a 1% (w/v) BSA solution at 280 nm. Would it be possible to
measure this absorbance on a modern UV-Visible spectrophotometer? (2 pts)
Calculate the absorbance at 280 nm for a 1 M BSA solution (BSA MW is 66 kD, 66,000 g/
mol). This value is the molar extinction coefficient (). How is it possible that another 66 kD
protein could have a very different molar extinction coefficient than BSA? (2 pts)
References and Appendix (4 points):
Cite three references on protein measurement from the peer-reviewed literature.
Attach the copies of your lab notebook pages showing absorbance data, calculations for
dilutions, and other observations.
25
Electrophoresis of Serum Proteins

Using SDS-PAGE
Electrophoresis is the movement of charged molecules through a
solid substrate that is mediated by the application of an electric
field. Electrophoresis is an important method of separating
biological molecules because it is highly sensitive to differences in
charge, mass, and shape and can separate very large molecules,
including proteins, which are the focus of this exercise. As the
characteristics of each protein affects how it behaves during
electrophoresis, a brief description of these qualities will first be
given:
Charge: The net charge on different proteins varies widely and is
due to a combination of the amino acid composition of the protein
and the pH of the environment. Acidic amino acids (aspartic and
glutamic acid) have a negative charge at physiological pH, while
basic amino acids (lysine and arginine) each have a positive
charge. Histidine residues, which a have pKa of 6.0, are neutral at
pH values above 7.0, but are positively charged (or partially
positively charged) at lower pH. However, most proteins have a
combination of acidic and basic amino acids, which partially
counteract each other.
Mass: Just as the net charge varies significantly from protein to
protein, the mass of proteins is also disparate. The smallest
proteins contain only about 20 amino acids while the largest known
protein, Titin, is composed of 38,138 residues 2. Given the average
molecular weight of amino acids of 110 Da, this give Titin an
approximate mass of 4,195,180 Da (~4.2 MDa)! It is important to
understand where this value of 110 Da/ residue comes from. If one
takes the average molecular mass of each of the 20 amino acids,
the average molecular weight is ~135 Da.
However, smaller amino acids such as
alanine and glycine tend to be more
abundant in proteins compared to large
residues, like as tryptophan and histidine.
This weighted average results in the actual
average weight being closer to 128 Da.
Additionally; an equivalent of one water (18
Da) is eliminated from each amino acid
during peptide bond formation (shown on the
right), resulting in the average molecular
weight of 110 Da. The loss of water during
pKa is the pH at
which one-half of
an acidic proton is
dissociated.
Daltons (Da) is a
unit of mass that is
equal to 1.0 g/mol.
26
peptide bond formation is shown in the figure on the right, where a

leucine residue is being added to the C-terminal end of the
tripeptide Ala-Val-Gly.
Shape/size: It is important to note here that the absolute threedimensional size of any native protein also depends on how tightly
packed it is. Large molecules, such as proteins, take on distinctive
shapes with widely varying length-to-width ratios. The three
dimensional shapes are maintained by hydrophobic interactions,
electrostatic interactions and disulfide bonds. Most cytoplasmic
proteins are globular and have generally round shapes, while
fibrous proteins are elongated.
Secondary
structures include
-helices and sheets. Tertiary
structures are
determined by how
these secondary
structures fold onto
each other.
Returning to electrophoresis, each of these factors can determine

the rate of migration through a solid substrate. The solid substrates
used for electrophoresis include materials such as paper, agarose,
and polyacrylamide. The polymerization of acrylamide and bisacrylamide creates a gel with pore sizes that can be varied by
changing the ratios of these reagents. These pores retard the
migration of proteins through the gel in a manner dependent upon
both size and shape. Separation of proteins by polyacrylamide gel
electrophoresis (PAGE) depends on differences in the frictional
coefficients of the proteins in an electric field and is a function of
size, shape, and charge. The rate of movement or velocity (v) of a
molecule within an electric field is a function of the net charge (z)
and frictional coefficient (f) of the molecule and the field strength (E)
of the system, thus giving the crude mathematical relationship:
v = Ez/f
There are many applications for which it is necessary or preferable
to separate proteins based solely by mass, rather than a
combination of charge, mass and shape. Achieving separation only
by mass is easily accomplished by the inclusion of two chemicals.
The first of these is -mercaptoethanol, a powerful reducing agent
that breaks disulfide bonds. This helps to both separate the
individual components of multipleMFQPAPKRCF TIESLVAKDS PLPASRSEDP IRPAALSYAN
subunit proteins and to denature
SSPINPFLNG FHSAAAAAAG RGVYSNPDLV FAEAVSHPPN
individual polypeptides. The second
PAVPVHPVPP PHALAAHPLP SSHSPHPLFA SQQRDPSTFY
PWLIHRYRYL GHRFQGNDTS PESFLLHNAL ARKPKRIRTA
reagent is sodium dodecyl sulfate
FSPSQLLRLE HAFEKNHYVV GAERKQLAHS LSLTETQVKV
(SDS), a negatively-charged
WFQNRRTKFK RQKLEEEGSD SQQKKKGTHH INRWRIATKQ
detergent that binds to proteins
ASPEEIDVTS DD
This 252 amino acid protein has 22 acidic (red) and
evenly along the polypeptide in a ratio
32 basic (green) residues (net = +10). If it were
of one SDS molecule per two amino
decorated with SDS, it would also have ~126
acids. This even decoration of
additional negative charges (net -116).
polypeptides with SDS results in the
27
masking of the intrinsic charges from acidic and basic amino acids
and makes the entire polypeptide negatively charged, while the
detergent action of SDS simultaneously denatures the protein by
disrupting hydrogen bonding within the protein. The sum of these
two actions of SDS, along with that of -mercaptoethanol, results
in the complete denaturation and linearization of the protein.
Electrophoresis performed in the presence of SDS is called SDSPAGE and resolves proteins according solely to their lengths. The
distance a particular protein migrates through a gel (Rf) is
inversely proportional to the log of the molecular weight (MW):
Rf = 1 / logMW
Once the proteins have been resolved by electrophoresis, the
separated proteins may be visualized in numerous ways, although
they are most frequently visualized by staining with chemicals or
specific dyes, as you will do in the following exercise. For different
applications, gels are not stained but are rather used as a
preliminary step in more advanced experimental procedures such
as western blots. It is also possible to purify specific bands of
proteins from polyacrylamide gels. Proteins purified from gels can
be used for polypeptide sequencing, mass spectroscopy (for
identification), or for other purposes.
Each of the compartments of an electrophoresis apparatus contains
buffer and an electrode. The solid support forms a bridge between
the two compartments through which charged molecules will move
when a potential (voltage) is applied. When the size of a particular
component of a sample is going to be determined, a separate
sample containing a mixture of molecules of known sizes may be
resolved by electrophoresis along with the samples; these are
frequently referred to as either markers or ladders. Additionally,
charged dye(s) that move through the electric field at a known rate
may be used to monitor the progress of electrophoresis in real time.
The situation at hand
When blood coagulates, the clear material that remains is serum,
which contains a mixture of many different types of proteins that
perform a wide range of functions. In this experiment you will use
SDS-PAGE to (1) separate the major protein components of human
blood serum, (2) determine the actual concentration of one of these
proteins in the serum, and (3) determine the size of this protein.
Ninety-five percent of serum proteins fall into five main groups and
are classified according to their mobility in an electrical field under
-mercaptoethanol
is a reducing agent
that breaks
disulfide bonds.
Dithiothreitol
(DTT) also can be
used for this
purpose.
28
standard (nondenaturing) conditions. From

highest to lowest mobility, they are albumin,
alpha-1 (1), alpha-2 (2), beta (), and gamma
() globulins (Figure 1). The 1, 2, , and globulins are each composed of groups of
related proteins; -globulins have an especially
wide range of higher molecular weights. Each of
these classes of protein, apart from albumin, are
composed of multiple polypeptide chains of
various lengths.
Figure 1. Schematic of the mobility of

the major classes of serum proteins, when
separated by paper electrophoresis
(nondenaturing conditions). Albumin has
the highest mobility, while gamma
globulins are the least mobile. Adapted
from1.
In the following experiment you will use SDSPAGE to (1) separate the protein components of
human blood serum, and (2) determine the
concentration of albumin in the serum by
comparing it to a series of serially diluted samples of bovine serum
albumin (BSA). The serially diluted samples will be used to create
a standard curve of protein, to which the human albumin band will
be applied in order to quantitate the amount of albumin. You will
also (3) determine the molecular weight of the albumin band. This
is the most common use of SDS-PAGE and can provide a good
approximation for the linear length of the polypeptide.
Serial dilution
means that you
dilute a sample,
and then use the
diluted sample to
make an even
more diluted
sample, etc.
29
Experimental Procedure
Part 1: Loading, running, and staining the gel
Caution: Take note of the warnings that are listed in the righthand margin.
Note: Pairs of students will share one gel, and each pair of
students will share an electrophoresis apparatus with another
group of two.
Step 1: Prepare the Ready Gel (Bio-Rad 7.5% polyacrylamide in
Tris-HCl buffer): Remove the gel from the package and
place it on a paper towel with the shorter plate facing up.
Use a razor blade to cut the tape along the black line
across the entire bottom of the gel cassette. Pull the tab
diagonally up toward the comb to remove. The tape must
be completely removed from the bottom of the gel before
the gel will make contact with the buffer in the lower buffer
tank; if a sticky film is still in place, use the razor blade the
scrape it off. The tape should be left on the sides of the gel
as this will hold the plates and gel together.
Step 2: As demonstrated by your instructor, assemble the
electrophoresis apparatus. Fill the inner reservoir between
the two gels with 1X Tris/glycine/SDS buffer and then fill
the outer reservoir until the buffer comes into contact with
the bottom of the gel (the slit that was exposed by
removing the tape). Once the buffer has been added,
remove the comb from the gel by positioning your thumbs
on the ridges on each end of the comb and pushing
upward slowly with a smooth, continuous motion. Be
careful to not pull the comb too quickly, as the resulting
suction can collapse the wells.
Wear gloves
when handling
polyacrylamide
gels.
Unpolymerized
acrylamide is a
potent
neurotoxin.
Read this step

carefully! If you
take the tape off
the sides of the
gel, it will fall
apart.
Step 3a: Prepare the serum sample in microcentrifuge tubes by

mixing the serum proteins (already diluted 10-fold) as
shown in Table1a.
Table 1a. Sample preparation for the human serum.
Sample
Description (dilution)
Human blood serum (1:10)
Human blood serum (1:10)
Sample
volume
10 L
10 L
dH2O
volume
0 L
10 L
Loading dye
volume
10 L
20 L
Total volume
(final dilution)
20 L (1:20)
40 L (1:40)
30
Step 3b: Prepare the BSA standards according to Table 1b. Start
by labeling microcentrifuge tubes with the final
concentrations or amounts. Dispense the appropriate
volumes of water into these tubes, then add the BSA. Mix
by pipetting slowly up and down three times, making sure
that you do not blow bubbles out of the pipette tip. Do
not vortex. Begin by preparing the most concentrated
sample (3.2 g/L) and then use this to prepare the 1.6
g/L sample. Continue preparing the serial dilutions until
all have been prepared. Then label a second set of tubes
with the amounts of BSA (16 g down to 1 g). Transfer
the appropriate amount of diluted BSA into these tubes
and then add 10 L of loading dye to each. Note: the
volume total column in Table 1b refers to the volume in
these samples before you transfer some to the next serial
dilution tube (not the final volume you should have).
Make sure to
wear your
gloves - the
running buffer
contains
methanol.
Table 1b. Sample preparation for the protein standard curve.

volume
dH2O
volume
BSA
volume
total
[final
BSA]
In a separate set of tubes

prep your samples
for loading on the gel
27.2 L
12.8 L
40 L
3.2 g/L
none
3.2 g/L
20 L
20 L
40 L
1.6 g/L
10 L + 10 L of loading dye
1.6 g/L
20 L
20 L
40 L
0.8 g/L
0.8 g /L
20 L
20 L
40 L
0.4 g/L
0.4 g/L
20 L
20 L
40 L
0.2 g/L
0.2 g/L
20 L
20 L
40 L
0.1 g/L
source of BSA
10 mg/ml stock
(1.00%)
Step 4: Check the level of the buffer between the gels: it should
still be up to the top of the glass plates. If it has leaked a
little, refill with 1X Tris/glycine/SDS buffer. If the buffer has
leaked a lot, ask your instructor to inspect your apparatus.
Load samples into the wells of the gel by positioning the
pipette tip loaded with the correct amount of sample and
loading dye over the top center of a well. The inner plate
(in contact with the inner chamber) of the ready gel is
shorter than the outer plate. This allows for a ledge to
gently rest the pipette on while dispensing the sample into
the well. The sample must be dispensed slowly into the
well in order to allow displacement of the buffer without
swirling and overflow. Note: the molecular weight (MW)
markers already are mixed with loading dye. Also, if you
change the samples loaded into any of the wells, make
sure to make a note of the change(s) in your notebook.
Having a hard
time loading the
lanes? Make
sure you took
the comb out.
31
Table 2. Sample volumes to load on the gel.

well #
1
2
3
4
5
6
7
8
9
10
volume of protein sample to load

5 L Human blood serum sample (1:40)
empty
20 L MW standards
20 L 0.1 g/L sample (1 g BSA)
group
member
1
1
1
1
2
2
2
2
2
Step 5: Place the lid onto the apparatus and plug the leads into
the power supply. Run the gel at a constant 200V for ~35
minutes. Every 5 minutes, check that the buffer in the
inner reservoir has not leaked and that the samples are
running fairly evenly from lanes 1 to 10. Shut off the
electrophoresis power supply once the blue tracking dye
has migrated off the gel.
The current
used for
electrophoresis
is dangerous.
Take the
appropriate
precautions.
Step 6: Stain the proteins in the gel as follows: Pour Coomassie

blue-250 staining solution into the plastic storage box (in
your drawer) to a depth of approximately 1.0 cm. While
wearing gloves, remove the gel from the Ready Gel
Cassette: pull off the tape from the sides of the plates and
then gently use a razor blade to pry apart the two plates.
Then, float the gel off of the plate by inverting the gel and
plate in the staining solution and agitate gently until the gel
separates from the plate.
Make sure to
wear your
gloves - the
Coomassie stain
contains
methanol.
Step 7: Place the lid on top of the storage box (leave it loose).
Place the box in the microwave and heat the gel for 1
minute. Place the box on the orbital shaker and allow the
dye to penetrate the gel for an additional five minutes.
During this time, wash the electrophoresis apparatus with
warm tap water and rinse 3 times with DI water. Set the
apparatus on paper towels on the lab countertop to dry.
Warning: the
staining solution
will be hot after
it has been
microwaved.
Step 8: After staining for an additional 5 minutes (wearing gloves)

carefully press the gel to the bottom of the box while
pouring the staining solution back into the original staining
32
solution bottle (gently place a finger on top of the gel to

keep it from slipping into the bottle).
Step 9: Add approximately ~10 mL of destaining solution (50%
MeOH, 10% Glacial Acetic Acid) to the gel in the box and
place the box on the orbital shaker for 3 minutes. Carefully
pour off the destain solution into the waste container, add
~20 mL of destain solution to the box and place it on the
shaker for 10 minutes. Repeat this destain step. Then,
pour off the destaining solution into waste and add 50 mL
deionized water, and place it on the benchtop for 10 min. If
the gel is still too dark (has a dark blue background making
it difficult to see the protein bands), pour off the water and
add ~20 mL destain solution again for 10 mins. Then
repeat with ~50 mL deionized water. Allow at least 30
minutes for the gel to equilibrate with dH2O, before you dry
it in cellophane. Set up the gel for drying in cellophane as
described next.
Make sure to
wear your
gloves - the
destain solution
contains
methanol.
33
Drying polyacrylamide gels

in cellophane
Step 1: If your gel is in destain, exchange the destain solution with
distilled water and allow the gel to equilibrate for 15
minutes before proceeding (the gel will expand a little bit).
Use water and paper towels to clean your bench area so
that it is free of dust and debris. Dry the bench.
Step 2: Unroll the cellophane onto the cleaned bench area and
place two gel drying frames, approximately 1 inch apart
over the cellophane. Cut the cellophane with a razor blade
or scissors, leaving 1-2 inches of excess cellophane above
and below the frames. Put the remaining roll of cellophane
away. Set the cut piece of cellophane to the side.
Step 3: Use the squirt bottle filled with deionized water to
completely wet an area of the bench slightly larger than
the cut piece of cellophane then gently place the
cellophane on the wetted area. Gently flatten the
cellophane out on the wetted area and push out all of the
bubbles from underneath the cellophane.
Step 4: Pour the polyacrylamide gel, along with some of the
water, onto the bottom half of the cellophane. Place a
frame over the bottom half of the cellophane and gently
nudge the gel into the center of the frame. Push any
bubbles out from beneath the gel. Remove the gel frame.
Step 5: Grab the top of the cellophane and drag it down over
the gel so that the top of the cellophane is even with
the bottom edge. The gel should now be sandwiched
between two layers of cellophane. Push air bubbles
out from between the two layers of the cellophane.
Step 6: Place a piece of the frame above the cellophane
and drag the cellophane-gel sandwich over the
frame.
34
Step 7: Place the second piece of the frame above the sandwich.
Step 8: Gently lift the frames and gel and use four binder clips to
hold the ensemble together. The two binder clips on the
bottom of the ensemble should be facing down, so that
the handles of the clips can be used to hold the gel in a
vertical position. Place the gel in the fume hood overnight
so that it can dry completely.
Step 9: Once the gel is dry, remove the clips and use a pair of
scissors or a razor blade to cut off the excess cellophane.
Make sure to leave between and 1 inch of cellophane on
each side of the gel. The gel can be stored indefinitely in
this manner and you can use a flatbed scanner to obtain
an image of the gel (as a .tif or .jpg file).
35
Analyzing the gel using the

ImageJ software
Part I: Quantitating the concentration of human albumin
Image J is free software from the National Institutes of Health3.
You may download this software on your personal
computer to do the analysis at home or you may use
the third floor university computer lab in the science
building where the software is already downloaded.
Step 1: Open the image file of the gel in ImageJ 3. Inspect the
image, in particular the lane from the standard curve with
the greatest amount of protein and the human serum lane
that you intend to analyze (the first serum lane was used in
the example, but you may wish to use the lane with less
protein). Use the rectangular selection tool to draw a box
around the wider of these lanes. Most likely, it will be the
lane containing the serum proteins.
Step 2: Once you have made the box, use the left and right arrow
keys to move the box over the lane containing the least
amount of BSA, and press the 1 key at the top of the
keyboard the number pad does not work for this function
(the results of this action are shown in Figure 2).
Figure 2. Selection of the first lane to be used for the protein

quantitation analysis. The lanes are numbered above the
gel and reflects the contents as shown in Table 2. Lanes
6 is used because it has the lowest amount of BSA. The
1 button is used to instruct the program to use this as
the first lane for analysis (bound by a green box).
Although you
can rotate the
image within
ImageJ, it is
easier if the
scanned image is
initially straight.
The rectangle
selection tool:
36
Step 3: Use the right arrow key on your keyboard to move the box
over the next lane and press the 2 key. Move the box
over the third lane and again press the 2 key. Continue
this process through each of the BSA standard curve lanes
and three more times for the lanes containing the serum
proteins that you wish to analyze (Figure 3). It is OK if the
boxes overlap, as long as each box contains albumin from
only one lane.
Figure 3. Selection of the other lanes for protein quantitation.

Lanes 6-10 (BSA) are selected first, followed by the
lanes with the human serum (1-4). Lane 5 contains MW
markers. For the lanes containing BSA, it is ok if the
boxes overlap, as long as each box contains the protein
for only one lane. The bracketed region (*) contains
additional immunoglobulin components of the human
serum, while the other bracketed region (#) contains
contaminants that we will see in future steps.
Step 4: Press the 3 key to generate the surface plots for the
various lanes. The plots for the various lanes will appear
separately, with lane #1 on top and the serum proteins at
the bottom. You can use the scroll wheel on your mouse to
move up and down through the various plots.
37
Step 5: Use the line selection tool to draw horizontal lines through
the bottom of the BSA peaks (Figure 4). For simplicity, you
can ignore the smaller peaks that result from the upper
(high molecular weight) bands in the lanes with the greater
amounts of BSA (seen on the left of the plots). For the
samples containing human serum, you will also need to
draw vertical lines to separate the peaks from the adjacent
1 proteins (like you did in the previous exercise).
The line
selection tool:
Figure 4. Use the straight line tool to mark the bottom of the peaks (blue lines). The
region below the lines is background. Plots A and B represent the BSA plot
for lane 7 (2 g), both before (A) and after (B) adding the line to subtract for
background. Plots C and D show the same process for lane 10 (16 g), along
with the presence of contaminants (*). For the lanes containing human
serum: E (lane 2) and F (lane 4), a vertical line must also be used to separate
the albumin peak from the immunoglobulin (#) peaks.
Step 6: Scroll back up to the top plot and use the magic wand tool
to choose the regions in each peak. When you get to the
plot of the serum proteins, select the albumin peak. A
Results window will automatically be generated.
The magic wand

tool:
38
Step 7: Copy the data to an Excel spreadsheet. Change the first

column of data to represent the actual amount of BSA
loaded in each lane (Figure 5).
Figure 5. Plotting a standard curve of the pixel counts from the serial dilutions
of BSA. Use a spreadsheet (left) to generate the standard curve
(right). The line equation is then used to determine the amount of
protein in the band of albumin in the human serum samples.
Step 8: Use the standard curve to calculate the concentration of

the human serum albumin in units of g/dL (dL = 0.1 L), the
standard units used in medicine. Do not forget to
compensate for the fact that the serum was supplied to you
already diluted 1:10 and you diluted it more. See Table 1a
or Table 2 for dilution factors.
Part II: Determining the size of the human albumin

Step 1: Open the image file of the gel in ImageJ [File->Open] or
[Crtl+O].Rotate the image so that the lanes are vertical.
Step 2: Click Analyze-> Gels-> Gel Analyzer Options and make
sure the invert peaks box is checked. Then, using the
Rectangle tool, make a rectangle through the center of the
molecular weight marker lane. You do not need to select
the entire lane.
Step 3: Press the 1 key at the top of the keyboard. A 1 should
now appear in the box over the molecular weight marker
lane.
Step 4: Use the arrow keys on your keyboard to move the box over
the lane with the smallest quantity of human serum and
The rectangle
tool:
These steps
should seem
very familiar, as
they are almost
identical to the
first steps of
Part I.
39
press the 2 key. A 2 should now be present in the box

over the second lane.
Step 5: Press the 3 key. This brings up a plot representing each
lane. The plot for lane containing the molecular weight
standards is shown in Figure 6.
Figure 6. Plot of the molecular weight standards. The portion of the lane
selected is shown (yellow). The higher molecular weight markers
are positioned on the left hand side of the plot. The extras includes
unresolved low molecular weight markers and the loading dye. You
will not use this peak for subsequent analysis.
Step 6: Choose the point selection tool, then hold shift and click
the first peak (250 kDa). This will label the peak as 1. The
label may be difficult to see as it is small and yellow,
located near the lower right corner of the box. While still
holding the shift key, continue and click all of the peaks,
except the extras (Figure 7). It is best to click all of the
peaks in order as they are labeled sequentially. Next, scroll
down so that you can see the plot of the human serum
and, while holding the shift key, click the peak for the
albumin.
Figure 7. The marked peaks. The top is the molecular weights markers and the
bottom is the human albumin. Note that the extras peak was not
picked.
The point
selection tool:
40
Step 8: Once the peaks have been marked, click Analyze ->
Measure (or press M). A results table appears with the
corresponding x and y values (Figure 8). Copy and Paste
these values into Microsoft Excel.
Figure 8. The results table obtained from the plot of the molecular weight
markers. The x values represent how far the proteins ran. The other
data is unimportant for this analysis. Do not be concerned if some
of the columns (e.i. IntDen) do not appear in your results window.
Row number 7 represents the human albumin.
Step 9: You will now use the known values of the molecular weight
ladder to create a standard curve. Calculate the log10 of the
molecular weights and use these values for the standard
curve (Figure 9). Plot the values using a scatter plot. Right
click a data point and select Add Trendline. Check the
boxes for Display Equation on chart and Display Rsquared value on chart.
Figure 9. How to analyze the molecular weight markers plot data. Column A
is the known molecular weights of the markers. Column B is the
log10 of column A. Column C is the data from column X (Figure
8). Use columns B and C to make the plot shown on the right. Do
not plot the data point corresponding to the human albumin. The
line equation and the R2-value are shown at the upper right.
Instructors: the
analysis will be
even more
accurate if only
the bands of
similar MW are
used for this
standard curve
(37-75 kDa).
41
Step 10: The equation obtained from plotting the molecular weight
standards can then be used to determine molecular weight
human albumin band. Plug the number for your human
albumin band (X-value; 420 in this case) as y and solve the
equation for x. Take the antilog (10x) of this value to get the
molecular weight of the human albumin. For the example
given, this corresponds to a molecular weight of 72 kDa.
References
1.
2.
3.
Harmening, D. M., Clinical Hematology and Fundamentals of

Hemostatis. 2nd ed.; F.A. Davis Company: Philadelphia,
1992; p 657.
Bang, M. L.; Centner, T.; Fornoff, F.; Geach, A. J.; Gotthardt,
M.; McNabb, M.; Witt, C. C.; Labeit, D.; Gregorio, C. C.;
Granzier, H.; Labeit, S., The complete gene sequence of
titin, expression of an unusual approximately 700-kDa titin
isoform, and its interaction with obscurin identify a novel Zline to I-band linking system. Circulation research 2001, 89
(11), 1065-72.
ImageJ, 1.45; National Institute of Health: Bethesda, MD,
2012. http://rsbweb.nih.gov/ij/download.html
42
Informal Report Grading Rubric:

Electrophoresis of Serum Proteins Using SDS-PAGE
Results (12 points):
(2 pts) Attach a printout of your gel. Label the lanes on this printout as
1-10. In the figure description, describe the contents of each lane.
(1 point) Show the table (from Excel) of your standard curve of the BSA
and the human albumin band.
(1 pt) Show the plot for your standard curve. Make sure you have the
axes labeled. Show the trend line, the R2 value, and the line equation.
(2 pts) Calculate the concentrations (of the original undiluted sample) of
the albumin in each of the human serum samples you prepared. Show
your calculations.
(2 pts) Use one of the concentrations of human serum albumin from the
previous question to calculate the total amount of albumin in a typical
person (2.8 liters of serum).
(2 pts) Show your table (from Excel) of your standard curve of the
molecular weight markers and your estimation of the size (in kDa) of the
human albumin band.
(1 pt) Use your estimated MW to calculate the number of amino acids in
the human serum protein using the average MW of all amino acids
which is 110 g/mol.
(1 pt) Using your estimated MW, calculate the molarity of the albumin in
the human serum sample.
(2 pt) What three properties of biological molecules influence the
mobility of the molecules in polyacrylamide gel electrophoresis (PAGE)?
(2 pt) SDS (in SDS-PAGE) eliminates two of these three factors.
What are these two factors and how does SDS eliminate them?
(1 pt) 2-mercaptoethanol is an ingredient in the loading dye. What
affect does it have on proteins?
(2 pts) You calculated the concentrations for each of the three human
serum samples you prepared. Are the values the same for each of
these samples? If not, provide a scientific explanation.
(2 pts) At the right is a list of the proteins that make up the markers.
Did you see all of the proteins in this list? If not, explain why. Hint: go
to the Bio-Rad website (or other website) and find the sizes of
proteins that are resolved on your gel (7.5% polyacrylamide).
(2 pts) Look up the number of amino acids in mature human serum
albumin. Compare the number of amino acids you calculated to the
reported number of amino acids you looked up. If there is a significant
difference, provide an explanation.
(2 pts) You used bovine serum albumin as a standard for human serum
albumin. Do you think this was appropriate? Could you use this as a
standard for any protein? Provide a rational for your answer.
MW Markers
250 kDa
150 kDa
100 kDa
75 kDa (pink)
50 kDa
37 kDa
25 kDa (pink)
20 kDa
15 kDa
10 kDa
43
Restriction Enzyme Analysis of Circular DNA

Plasmid DNAs are small, circular DNA molecules that exist naturally in some species of
bacteria. Plasmids duplicate independently of chromosomal DNA and are generally not
required for normal cellular metabolism. The small size of plasmids allows them to be
easily handled in a laboratory setting. In fact, a large percentage of biochemical and
biological research depends on cloning DNA fragments into plasmids for further study.
In this experiment, you will analyze a plasmid DNA sample by restriction endonuclease
digestions.
Before todays experiment, you need to have some understanding of this special family
of enzymes that are used in recombinant DNA work. These enzymes are called
restriction endonucleases (restriction refers to the fact that these enzymes are used to
digest foreign DNA often viral to restrict the growth of the invaders; endonuclease
refers to the fact that these enzymes cut in the middle of DNA sequence endo and
are present in the nucleus). The figure below shows the general mechanism through
which restriction endonucleases work. Basically, they recognize specific sequences that
have the special characteristic of being palindromic, or having the same sequence in
both directions.
How restriction endonucleases work:
These enzymes recognize palindromic
sequences (shown in color; the direction of
the repeat sequence is indicated by the
arrows). The enzyme in this case EcoRI
recognizes this sequence and cuts in a
staggered manner to generate two
fragments. These staggered ends are called
sticky ends.
In the following experiment, you will analyze a circular DNA that you have digested with
different combinations of restriction endonucleases. One of these DNA samples will be
digested with the restriction endonuclease HindIII (as outlined in the figure on the next
page). The recognition sequence for this restriction endonuclease is present in three
positions in the plasmid; digestion of this plasmid with HindIII therefore results in the
generation of three DNA fragments. The size of these fragments can be determined by
separating the DNA pieces on an agarose gel (similar to gelatin), which separates the
DNA fragments by size: small fragments migrate through the agarose gel more quickly
than do larger fragments.
44
Figure 1. Digestion of plasmid DNA

with a restriction endonuclease:
(Top) a map of plasmid pGBKT7. The
red one (1) indicates the position of
nucleotide 1 (arbitrarily assigned). The
nucleotide positions of the HindIII
restriction sites are listed in black,
while the distance between these sites
is shown in blue. Digestion of the
plasmid with HindIII results in three
cuts and therefore three DNA
fragments (the position of nucleotide 1
is present in the fragment having a
length of 1498 nucleotides).
(Bottom) a picture of an agarose
electrophoresis gel with bands of the
plasmid DNA after being digested with
HindIII. The first lane has a marker
with known nucleotide lengths (in base
pairs, bp) listed on the left, while the
second lane has the digested plasmid.
Note how the smaller fragments
migrate further towards the bottom of
the gel.
In the following experiment, you will digest a DNA plasmid (pGBKT7) using several
different combinations of restriction endonuclease enzymes. You will then estimate the
position(s) of the sites that the unknown restriction enzymes cut. The unknown enzymes
will cut, either once or twice, in the middle of one of the three fragments (between two of
the HindIII sites), resulting in the production of four (or five, if it cuts twice) bands. You
will then determine where the unknown enzymes cut.
45
Procedure
Caution: Wear gloves when handling ethidium bromide or gels containing
ethidium bromide. This chemical intercalates between DNA bases and fluoresces
in UV light. It is a known mutagen.
Waste: The buffer can be poured down the sink and the agarose gel can be
disposed of in the trash.
Part 1: Digesting plasmid DNA

You will work in groups of three. There are four restriction endonucleases (three
different unknowns) in the freezer. Each member of the group will use HindIII and one
unknown. No two members of the group should have the same unknown number. Each
group member will set up three DNA digestions: one with HindIII alone, one with the
unknown enzyme alone, and one using both enzymes simultaneously.
Step 1: Use the information in the next paragraph to finish filling out the following table:
Table 1. Components of the DNA digest reactions.
(--------------------------Volumes-------------------------)
Reaction
Reaction #1
Reaction #2
Reaction #3
Component
pGBKT7 2 L
2 L
2 L
10X buffer 2 L(
)
2 L(
)
2 L(
)
0.25% BSA 2 L
2 L
2 L
Enzyme #1 2 L(
)
2 L(
)
2 L(
)
Enzyme #2 _ L(
)
_ L(
)
_ L(
)
dH2O __L
__L
__L
Total volume 20 L
20 L
20 L
Reaction numbers 1 and 2 are known as single digests because only one enzyme is
used to cut the DNA. In Reaction #1 use only HindIII (write HindIII in the parentheses
for enzyme #1), and put a zero for enzyme #2 volume. Calculate the volume of dH2O
needed to make the total volume of Reaction #1 equal to 20 L. In Reaction #2 use
only your unknown enzyme (write the unknown number in the parentheses for
enzyme #1), and put a zero for the volume of enzyme #2. Reaction number 3 is a
double digest because two enzymes are used to cut the DNA. For reaction #3, use
46
2 L of HindIII and 2 L of your unknown. Fill out the table above with these volumes
and enzymes. Use Table 2. below to choose the best buffer for each digest and write
the number of the buffer in the 10X buffer parentheses in Table 1. Use the completed
Table 1. as a guide when adding the each reaction component to the individual
microfuge tubes.
Table 2. Best buffer to use for each reaction.
Single digest
HindIII
unknown #1
unknown #2
unknown #3
2
2
4
3
+ HindIII
Double digest
2
2
2
Step 2: Label three microcentrifuge tubes with your initials and the number of the
corresponding reactions. For instance, Todd Krolls first tube would be labeled
TK#1. You may write more details on the side of the tube if you wish. BSA is an
enzyme stabilizing agent. Completely thaw and mix the buffer(s) and the BSA
that you need before adding the restriction enzyme. Mix by slowly pipetting the
entire volume up and down within the pipette tip. Be careful to NOT blow air
out of the pipette tip so that bubbles do not form in your solution.
Always KEEP ENZYMES ON ICE when they are not in the freezer.
Step 3: Begin pipetting! Dispense the appropriate volumes of the reaction components
(in the first table) into the appropriate microcentrifuge tubes. Add the dH2O first,
and then start at the top of the list and add each component with gentle mixing.
Mixing should be accomplished by gently pipetting up and down. Try to get as
few air bubbles in the mixture as possible, as air bubbles denature protein (the
restriction endonucleases are protein!).
Step 4: When you have assembled the digestion reactions, place the microcentrifuge
tubes in a flotation device in a 37 C water bath. Allow the digests to go for at
least 45 minutes, with an hour being preferred.
Part 2: Preparing, Running, and Analyzing the Agarose Gel

Step 1: Prepare an agarose gel. One group of three students will share a single
agarose gel and you should all work together to prepare it. You will prepare a
45 mL volume gel that contains 1% agarose in Tris-Acetate-EDTA (TAE) buffer.
Recall from general chemistry that percentages are sometimes used to prepare
solutions. The calculation for preparing a 40 mL 1 % agarose gel is as follows:
47
thebasicequation
putinyourknowns
solveforgramsofAgarose
Weigh out the appropriate amount of agarose and put it into a 250 mL
Erlenmeyer flask. Add 45 mL of TAE buffer.
Step 2: Now, microwave the agarose and TAE for 1 minute and watch it to be sure it
does not boil over. If it begins to boil, shut off the microwave. When the
microwave is done, use a hot hands to remove the flask. Gently swirl the
mixture, being careful to not allow the agarose to boil over and burn you. If you
see any undissolved bits of agarose still floating (you probably will) then
microwave for an additional 15 seconds. Check the solution again and repeat
the microwaving until the agarose is completely melted.
Step 3: Reread the cautions at the beginning of the procedures section! Add 2 L
of ethidium bromide solution and swirl to mix the solution. Allow the agarose to
cool until you can hold the bottom of the flask in your bare hand for 5 seconds.
While you are waiting for the agarose to cool, place the gel tray in a clamping
frame in the electrophoresis apparatus and place a 15-well comb in the
notches. Two trays can fit in one clamping frame.
Step 4: Once the agarose has cooled sufficiently, pour the agarose into the tray (until it
is 2-3 mm from the top of the fingers of the comb). Wait for the gel to solidify
this will take approximately 10 minutes. While your gel is solidifying, obtain your
DNA samples from the water bath (if at least 45 minutes have elapsed, if it has
not been 45 minutes then you need to wait!) and the DNA marker (one per
group). The DNA samples will contain approximately 20 L of DNA. Add three
48
L of DNA loading dye to each of these samples. The marker DNA already has
loading dye added; if not, check with your instructor.
Step 5: Once your gel has solidified, carefully remove the tray(s) (with gel and comb)
from the clamping frame and put it in the electrophoresis apparatus. Make sure
that the gel is correctly oriented for the DNA samples to migrate towards the
anode. Fill the electrophoresis reservoir with TAE buffer until the gel is
completely covered. Remove the comb carefully. Load your DNA samples
(~23 L) into the wells in the gel along with the DNA standards or size marker
(~12 L). Do not worry if you cannot load the entire DNA sample into the well.
Make sure you take note of what is in each lane of the gel! Use the Figure
2. below as a guide for loading the gel.
Figure 2. Map of the electrophoresis gel showing lane contents.
Step 6: When all of the samples have been loaded, place the lid on the top of the
apparatus and connect the leads to the power supply. Turn on the power
supply and turn it up to 110 V. Press the button with a picture of a running man
to start the gel. Let the gel run for 30 minutes. This is a good time to review
electrochemistry.
Step 7: Turn off the power supply and (wearing gloves!) remove your gel from the
apparatus. Hold your fingers over the ends of the gel so that it does not slide off
the tray and break into a million pieces on the ground. Nothing is more
frustrating than reassembling a million pieces of agarose gel.
Step 8: Have your TA or instructor help you to take a picture of your gel.
49
Step 9: Compare the banding patterns between the different lanes of the gel. You
should see three prominent bands in the plasmid digested with only Hind III
(similar to that shown in the Bottom of the Figure 1). Do you see four bands in
the other DNA samples? Determine in which of the DNA fragments each of the
other restriction endonucleases cut.
Figure 3. Image of a sample of 1 kb marker DNA after

electrophoresis. Note that the 3.0 kb band is extra bright.
Unless you run the gel for a long time, you will probably not
be able to see separation of the larger bands. It is also
possible that you will not be able to see the smallest band.
This is taken from the New England BioLabs website.
50
Grading rubric: Restriction Enzyme Analysis of Circular DNA

(4 pts) Attach the picture of your gel to a piece of paper from your lab notebook
and number each of the lanes. On the paper to which the picture is attached, list
the contents (DNA and enzymes) in each lane.
(6 pts) Redraw the diagram for the circular plasmid DNA, including the restriction
endonuclease cleavage sites for HindIII and each of the three unknowns.
(5 pts) Explain how you determined where the unknown enzymes cut the plasmid
DNA.

(2 pts) Were there any bands on your gel that you could not account for? What
could lead to unexpected banding patterns?
(2 pts) Why were different buffers used in this lab and what is the purpose of the
BSA? Use the search dialog box at the New England BioLabs website for help.
(2 pts) Explain why ethidium bromide can be used to locate bands of DNA on the
gel. Discuss the precautions you took while using this chemical.
(2 pts) The bromophenol blue dye in the loading dye solution migrates in the gel
at an apparent size of 400 to 500 bp while its MW is actually much smaller. What
physical properties of the bromophenol blue could account for its electrophoretic
behavior?
(2 pts) If you wanted to insert a gene into a plasmid, why would it be helpful to
know the plasmids restriction enzymes cleavage sites?
Extra credit
(1 pt) Use a ruler, calculator and graph paper to calculate the size of the bands
made when HindIII is used. Use the separate instructions for Analyzing Agarose
Gel Electrophoresis of DNA (Appendix III). Compare the calculated sizes to the
sizes given in the introduction.
Note: please attach your yellow carbon sheets from your lab
notebook.
51
Isolation of an Enzyme: Acid Phosphatase

Biochemical preparations involve the isolation or purification of cellular components
(such as organelles), particles (ribosomes), or one of four major types of biologically
important molecules (protein, carbohydrate, nucleic acid, and lipid). Special laboratory
techniques including cell lysis, tissue homogenization, filtration, centrifugation,
chromatography, salt or solvent precipitation, and concentration can be employed. In
this experiment you will use some of these procedures to isolate the enzyme acid
phosphatase from wheat germ (Triticum aestiva).
Before continuing, it is important to differentiate between protein isolation (which you will
conduct here) and protein purification. Isolation is a generally more crude form of
purification in which a protein is separated from organelles, other types of
macromolecules, and from some of the other cellular proteins. Purification of proteins
involves a continuation of isolation process until the protein of interest has been
separated from essentially all other cellular proteins (in other words, the target protein is
the only component from the original cell remaining in the sample). Protein isolation is
often used when it is either (1) unnecessary to have all other proteins removed from a
preparation, or (2) detrimental to the protein (the protein becomes unstable) when all
other proteins are removed from the preparation. Steps 1-2 of the following flow chart
are generally used for isolation, while steps 3-4 (and sometimes 5 and/or 6) is used for
protein purification.
Flow Chart Depicting Typical Protein Isolation and/or Purification
1.
2.
3.
4.
5.
6.
Disruption and extraction of cells or separation of cells from a cell supernatant

Concentration of the extract or supernatant
a. Ultrafiltration
b. Organic solvent precipitation
c. Salt precipitation
i. Dialysis to remove excess salt or
ii. Gel filtration to remove excess salt
Chromatographic separation
a. Ion-exchange chromatography
b. Gel filtration chromatography
c. Affinity chromatography
Concentration of active components of the chromatographic fractions
a. Centrifuge concentrator
b. Lyophilization
c. Ultrafiltration
d. Polyethylene glycol dialysis
Preparative electrophoresis or isoelectric focusing (for certain applications)
Crystallization (for certain applications).
52
1. Disruption and extraction of cells. The starting material may vary widely,
depending on the goal of the study. For isolation/purification of endogenous protein, the
source of the desired component may be an intact organism or some specific part of a
plant or animal. If the protein to be purified is recombinant, then the source of the
protein is generally either E. coli or cultured cells harboring an expression plasmid
encoding the desired protein. Once the source of the protein is acquired, the first step is
usually lysis of the tissue cells to release the desired components. Animal cells have a
weak outer plasma membrane that is generally easy to lyse by osmotic shock, whereas
plant cells and bacteria are more difficult because they are protected by a tough cell
wall.
Different methods of lysing the cells are used to extract the internal components for
isolation. As previously stated, lysing of cultured animal cells is generally very simple as
the plasma membrane is easily disrupted by transferring the cells to a hypotonic
solution, causing the cells to burst by osmotic lysis. Other methods are often used if the
animal cells of from tissue, which often have tough connective material protecting the
cells. Similarly, more vigorous methods are used to break the tough cell walls of
bacterial, yeast and plant cells. A partial list of these methods include:
French press this method places the cells under extreme pressure and then
releases the cells through a small hole to the much lower atmospheric pressure.,
with the rapid decrease in pressure lysing the cells.
Sonicators - these produce ultrasound waves, which disrupt cell walls by
shearing and cavitation of the cell wall.
Bead mills are used to tear the cell wall from the cell by rapid vibration, similar to
sonicators.
Blender, grinding and homogenizers combine abrasive and lysing by osmotic
pressure to disrupt cell membranes.
Chemical means, such as strong base (NaOH, most frequently) are also utilized
to disrupt cells walls. This is the most frequent method used when DNA is being
isolated from bacterial cells.
It is necessary when making a cell extract that the tissue or cells are quickly suspended
in a suitable buffer that will stabilize the cellular component being isolated. After the
cells have been lysed, a clear extract is obtained by centrifugation and filtration. When
proteins are being isolated, it is often necessary to protect the proteins from proteases
(proteins that digest protein) by including protease inhibitors in the resuspension buffer.
2. Concentration of the extract. Centrifugation is the process of rapidly rotating a
receptacle containing a slurry of solid particles in a fluid; the particles are sedimented by
a greatly increased gravitational field. The centrifugal force or gravitational force (gforce) is proportional to the square of the angular velocity (expressed in revolutions per
minute) times the radial distance from the center of rotation. Successful centrifugation
depends on the time of centrifugation as well as the gravitational force developed. The
two main types of centrifugation include differential and isopycnic centrifugation:
53
Differential centrifugation separates cellular components by differences in

solubility. Centrifugation of total cell extract will precipitate the insoluble
materials, while leaving the soluble protein and nucleic acids in solution.
Changing the buffer conditions can allow for the specific precipitation of particular
proteins or nucleic acids based upon their chemical characteristics. An example
of this is salting out, which is describe later in this tutorial.
Isopycnic centrifugation separates cellular components by differences in their
densities. A gradient of sucrose (or other material) is made in a centrifuge tube
and the cellular components are gently transferred to the top of the gradient (low
density). Upon centrifugation at very high centrifugal force, the components
migrate down until their density matches the density in the gradient. This method
is often used to isolate different organelles or particles (such as ribosomes).
In addition to these basic centrifugation methods, various other methods of

concentrating samples are used in different situations:
Biological solutions are frequently dilute and contain thermolabile molecules,

which cannot be concentrated by heating the solution. Very large volumes,
10-100 L, can be concentrated by using membrane filtration, which utilizes a
semipermeable membrane that does not allow passage of the molecules of
interest. More recently, this method has been combined with centrifugation in
order to both speed up the process and make it more useful for concentrating
smaller proteins samples (1-100 mL).
A less frequently used method is the addition of a solid, insoluble material (such
as highly cross-linked Sephadexes and Bio-Gels) that has an affinity to absorb
water. Following hydration of the gel material, the slurry is filtered by aspiration,
resulting in a filtrate that has one-third to one-half the original volume. A much
higher concentration (10-100 fold) can be obtained by filling a dialysis bag with
the solution to be concentrated, placing it in a beaker or crystallizing dish, and
covering it with powdered polyethylene glycol (MW 20,000).
Proteins are often concentrated by precipitation. Most globular proteins fold so that they
have their hydrophobic amino acid residues in the core and their hydrophilic
(water-soluble) residues facing out from the core of the protein. This allows water to
interact with the hydrophilic residues and keep the protein in solution. Two methods can
be used to disrupt the solubility of proteins in their aqueous environment: (1)
precipitation by water-miscible organic solvents, and (2) salting out with a high
concentration of salt.
The two most commonly used organic solvents used to precipitate protein are ethanol
and acetone. The addition of these organic solvents to an aqueous solution of proteins
produces a reduction in the solvating power of the water for the charged, hydrophilic
protein molecule, leading to aggregation and precipitation of the protein. A feature
affecting the precipitation is the size of the protein; in general, the larger the protein
molecule, the lower the percentage of organic solvent required to precipitate it.
54
Precipitation also occurs at a lower organic solvent concentration when the protein is
near its isoelectric point. When precipitating a protein with an organic solvent, the
temperature should be maintained around 0o to prevent denaturation due to the heat of
mixing when the organic solvent is added to water. The protein containing extract and
the organic solvent should be added relatively slowly, with efficient stirring, to prevent
the formation of locally high concentrations of organic solvent. After the addition of the
organic solvent, the precipitate is removed by centrifugation and decanting of the
supernatant. The precipitate is then dissolved in a minimum amount of buffer.
Proteins also can be precipitated by salts, a process called salting out. Water solvates
the added salt ions, thus decreasing the availability of the water to keep the protein in
solution and exposing the hydrophobic areas of the protein. The exposed hydrophobic
regions then interact with each other to generate aggregates that precipitate. The
procedure involves dissolving the salt into the solution containing the protein, much like
the addition of an organic solvent. The most commonly used salt is ammonium sulfate,
which is very soluble in water. At 4C, saturated ammonium sulfate ((NH4)2SO4; MW =
132.14 g/mol) is approximately 4 M (or the addition of 717 g (5.4 moles) to one liter of
solution; this increases the volume significantly). The optimum concentration of
ammonium sulfate needed to precipitate a protein is empirically. A disadvantage of the
salting-out technique is that relatively large amounts of salt remain with the precipitate
and usually must be removed before the next step can begin. The removal of salt can
be accomplished by dialysis or gel filtration, but both procedures require an additional
step and usually lead to an increase in the volume of the protein solution.
Low molecular weight materials such as salts, and some biological materials, such as
amino acids, coenzymes, and low molecular weight carbohydrates, can be removed by
dialysis from macromolecular materials. Dialysis is the process of separating smaller
molecules from larger ones in solution by use of a semipermeable membrane that
permits passage of the smaller, but not the larger, molecules. Cellophane dialysis tubing
have pores that will not permit the passage of molecules having a molecular weight
exceeding a given size (for example, you will use dialysis tubing with a molecular weight
cutoff of 12,000-14,000 Da). Sufficient tubing should be used so that approximately half
of the tube volume is void when the sample solution is added. (Relatively high salt
solutions will take up water in the tube, and the expansion can cause the tube to break.)
After the sample solution has been added, the tubing is closed just above the top of the
solution, air is removed, and the tube is tightly sealed by either knotted at the end and
tied with a string or using specially-designed clamps. The bag is placed in a container
with 10 volumes or so of water or buffer. After 4-6 hours, an equilibrium is established,
at which point the concentration of the dialyzable material is the same outside and
inside the dialysis bag. This process is normally repeated one or more additional times
in order to completely exchange the initial buffer with the desired buffer.
In the following experiment you will isolate the enzyme acid phosphatase. Although this
basic procedure will not result in highly purified protein (which would require column
chromatography), the enzyme preparation will be pure enough so that the impurities will
not interfere with the activity of the enzyme. You will collect fractions during the isolation
55
process so that you can monitor the increase in specific activity. You will then use the
isolated protein for a series of experiments to characterize acid phosphatase. For this
final exercise, the protocol will be presented in paragraph format; this is akin to what
you will encounter when trying to repeat a published experimental protocol.
Reference: A reference for the procedure is Minch, Michael J., Experiments in
Biochemistry, Prentice-Hall, 1989.
Procedure
Part 1. Isolating the enzyme.
NOTE: It is important that at all times during this experiment you keep the
enzyme preparation ice cold. When the enzyme is stored for more than 3 hours
during the isolation procedure, store it frozen.
Preparation of Crude Extract. Grind 30 g of fresh, uncooked wheat germ in a cold
Waring blender in the cold cabinet for 1 min. Add 300 mL cold 0.3 M acetate buffer, pH
4, and homogenize in Waring blender for about 30 sec. Stir gently for 30 min on a stir
plate in the cold cabinet or in an ice bath on the lab bench. Centrifuge at 12,000 X g for
10 min at 4oC and discard the precipitate. Filter the supernatant solution with a funnel
and cheese cloth to remove suspended material and particulates. The resulting solution
is the crude homogenate. Measure and record the total volume, then remove a 10 mL
sample and freeze in a test tube that you have labeled with your name and crude
using label tape. Cover the test tube with parafilm.
Precipitation with Acetone. Add cold acetone (-20oC; stored in the freezer) to the
crude homogenate to a final volume of 55% Acetone (v/v). Set up an ice bath in the
hood on a stir plate. Place the crude homogenate in the ice bath and stir while very
slowly adding the acetone; continue to stir gently for 15 min. Decant if possible to
reduce the volume, but don't pour off solution containing significant turbidity. Centrifuge
the turbid solution at 12,000 X g for 10 min at 4oC to collect the precipitated protein.
Discard the supernatant solution in a labeled Acetone Waste container. Add 40 mL of
cold, distilled H2O and stir gently for 45 min on a stir plate in the cold cabinet or in an ice
bath on the lab bench to allow precipitated enzyme to dissolve.
WASTE: Discard the 55% acetone supernatant in the waste bottle stored in the hood.
Centrifuge the dissolved 55% acetone precipitate at 12,000 X g for 10 min at 4oC to
remove undissolved material. Discard the precipitate. Measure and record the total
volume, then remove a 10 mL sample of the supernatant, freeze in a test tube, cover
with parafilm and label it the "55% Acetone" fraction.
Precipitation with Ammonium Sulfate. You will precipitate proteins using two different
56
concentrations of ammonium sulfate. Put your sample in a 100 mL beaker and stir bar
on a stir plate in the cold box. Turn the stir plate on and set it so the bar is spinning
smoothly, but not fast enough that it creates protein-killing air bubbles. Use the
Ammonium Sulfate PrecipitationTable at 4 C at the end of this procedure section to
calculate the amount of solid ammonium sulfate to add to bring the solution to the first
(lower; e.g., 65%) of the two concentrations. Add the (NH4)2SO4 slowly to the sample,
allowing the ammonium sulfate to dissolve between additions. Once all of the
ammonium sulfate has been added, stir gently for 20 min, then centrifuge at 12,000 X g
for 10 min at 4oC. Keep both the pellet (precipitated protein) and the supernatant. Pour
the supernatant into a graduated cylinder to measure the volume. Resuspend the
protein pellet by stirring in 20 mL 0.3 M acetate buffer, pH 4; label this the " 65% AS"
fraction (represents the concentration of ammonium sulfate you used) and save for
dialysis (see below). Pour the supernatant into a clean beaker and, using your
determined volume, calculate the amount of ammonium sulfate needed to bring the
concentration up to your second (higher; e.g., 85%) concentration. Add the additional
(NH4)2SO4 slowly to the supernatant to achieve the final concentration and stir gently
for 20 min in the cold box. Centrifuge the sample at 12,000 X g for 10 min. Discard the
supernatant. Suspend the precipitate in 20 mL 0.3 M acetate buffer, pH 4; label this the
"85% AS" fraction and save for dialysis (see below).
Dialysis. Prepare two 25 cm strip of dialysis tubing by boiling for 30 min in distilled H2O
containing a pinch of Na2EDTA (a chelating agent that complexes metal ions). Then boil
for 10 min in distilled, deionized H2O without Na2EDTA. Transfer the tubing to distilled,
deionized H2O and store cold until ready for use. Fold over and tie closed one end of
the tubing with string and pour in one (NH4)2SO4 fraction. Fold over and tie closed the
other end, leaving space in the tubing for an increase in volume due to H2O entering the
tubing. Do the same with your other (NH4)2SO4 fraction. Use excess string to attach a
microcentrifuge tube to each end of each of the dialysis tubes this will help keep the
tubing afloat, so that it does not sink and hit the stir bar. You can keep track of which
(NH4)2SO4 fraction is which by labeling the tubes or tying different numbers of knots in
the string. Put both dialysis tubes in a 1000 mL beaker and add 800 mL of 0.3 M
acetate buffer, pH 4. Dialyze with gentle stirring for three hours (or overnight), then pour
off the buffer (down the sink) and put in fresh buffer and repeat dialysis for at least three
hours to reach equilibrium. Once dialysis is complete, untie the tubing and pour the
contents of each tube into separate labeled beakers. For each fraction, measure and
record the volume and then freeze in a test tube or beaker.
Use the frozen portions from each of the above fractions (the crude lysate, the 55%
acetone fraction, and the two ammonium sulfate fractions) to measure protein
concentration, enzyme activity, and specific activity as described below.
WASTE: The acetate buffer used in dialysis and the Na2EDTA solutions may be
poured down the sink.
57
58
Part 2.
Measuring protein concentration in the four enzyme
fractions.
Use the Biuret test. Run a series of microcentrifuge tubes with the following contents:
Protein
Assay
Standard
Curve
Table 1. Experimental set-up for determining protein concentration.

tube # sample
L sample L H2O
L biuret
mg protein Abs540nm
1 (use as blank)
1500
2
750
750
3
750
750
725
750
0.25 mg
4 10 mg/mL BSA 25
5 10 mg/mL BSA 75
675
750
mg
6 10 mg/mL BSA 125
625
750
mg
7 10 mg/mL BSA 175
575
750
mg
8
crude
250
1250
9
crude
250
500
750
10 55% acet
250
1250
11 55% acet
250
500
750
12 low% AS
250
1250
13 low% AS
250
500
750
14 high% AS
250
1250
15 high% AS
250
500
750
Recall that 10 mg/mL = 1000mg/100mL = 1g/100mL which is a 1% solution.
Mix the reagents in each tube thoroughly by vortex or by inverting them three times and
then warm the tubes at 37oC for 10 minutes and cool to room temperature (a room
temperature water bath will speed the cooling). Read the absorbances of each sample
at 540 nm with a UV-visible spectrophotometer, using the contents of tube #1 to set the
absorbance at 0.00. Subtract the average absorbance of tubes 2 and 3 from all tubes
containing biuret reagent (4-7, 9, 11, 13, and 15). Subtract any absorbance in tubes 8,
10, 12, and 14 from the absorbance in the following tube; this corrects for absorbance
due to turbidity in the protein fraction.
Use the corrected absorbances in tubes 4-7 to plot absorbance vs. mg protein. Tube #4,
for example, has 0.25 mg BSA (1.00% BSA is 10 mg/mL, and 0.025 mL (which is 25L)
x 10 mg/mL = 0.25 mg BSA) (the other mg amounts are left for you to calculate). From
this standard curve of corrected absorbances, determine (1) the concentration (mg/mL)
and (2) the total mg of protein in each enzyme fraction. To determine concentration,
remember that you used a total of 0.250 mL (250 L) of each fraction sample to
determine its protein content.
59
Part 3.
Measuring enzyme activity in the four enzyme fractions.
The reaction catalyzed by acid phosphatase that we will study is:
The substrate is p-nitrophenylphosphate (abbreviated PNPP) and the product is pnitrophenol (abbreviated PNP). In the presence of base, the p-nitrophenol (PNP)
product is ionized and its color changes to the yellow p-nitrophenoxide ion:
Since the other reactants and products are colorless, measurement of the reaction rate
is based on the amount of product formation, which is the amount of yellow color formed
after exposure to base.
The first step is to establish the linear relationship between absorbance and known
concentrations of p-nitrophenol. This is only done in order to prove to you this linear
relationship, described by Beers Law. Establish this relationship by making up test
tubes with the contents as shown in Table 2. Note: there is plenty of KOH (it is 1 M) in
the last tube to convert the PNP into p-nitrophenoxide (it is only 5.0 X 10-5 M).
Table 2. p-nitrophenol standard curve.
L 1.0 M KOH
tube # volume 5.0 x 10-5M PNP
1
0 L
1500 L (use as blank)
2
250 L
1250 L
3
500 L
1000 L
4
750 L
750 L
5
1000 L
500 L
6
1250 L
250 L
Abs405nm
__________
__________
__________
__________
__________
__________
Read the absorbances at 405 nm using a UV-visible spectrophotometer, setting the

absorbance to 0.00 with tube #1. Plot the moles of p-nitrophenol versus Abs405nm to
60
establish the linear relationship. Since you have verified that the relationship is linear,
you may use a simple proportionality to calculate product formation (see the enzyme
activity calculation on next page).
Inspect Table 3; tube #1 is used to blank the spectrophotometer, tubes #2-4 are used to
establish the ratio of moles PNP to Abs405nm; tubes 5-16 are to determine the activity of
each of your fractions. Label sixteen 1.6 mL microcentrifuge tubes in an appropriate
fashion. The "cocktail" solution (stored in the freezer) is composed of: 5 mM pnitrophenylphosphate (substrate), 0.1 M acetate, pH 5.2 (buffer), and 0.1% BSA (as an
enzyme stabilizing agent). Pipette the volumes of cocktail, water, enzyme, PNP and
KOH into the appropriate tubes as directed by Table 3.
Before you begin, it is important to note that you will start the reaction by adding
enzyme and stop it by adding KOH. Tubes that are used for correcting for absorbance
not from enzyme activity are generated by adding KOH before enzyme. The tubes used
for this purpose are bolded and marked with an asterisk in Table 3.
Table 3. Detail of experimental set-up for testing enzyme activity.
L PNP
L KOH
tube #
L cocktail
L H2O enz. fraction (L)
1
(blank)
1200
300
2
900
300
300
3
900
300
300
4
900
300
300
5*
900
290
crude homog. (10)
300*
6
900
290
"
300
7
900
290
"
300
8*
900
290
55% acet (10)
300*
9
900
290
"
300
10
900
290
"
300
300*
11*
900
290
low% (NH4)2SO4 (10)
12
900
290
"
300
13
900
290
"
300
14*
900
290
high% (NH4)2SO4 (10)
300*
15
900
290
"
300
16
900
290
"
300
*In asterisked tubes add 300 L KOH before adding the 10 L enzyme fraction.
Add 300 L of KOH to tubes 5, 8, 11, and 14. Add the PNP to tubes 2-4. Add water and
cocktail to the tubes indicated and incubate all 16 tubes at 37oC for about 5 min to bring
them to reaction temperature. After the tubes and their contents have reached 37oC
leave them in the water bath to maintain the reaction temperature at 37oC and add 10
L of the appropriate enzyme fraction by pipette into the solution in the tubes. After
adding the enzyme to each tube, be sure to mix the enzyme with the reaction
buffer which contains substrate by inverting the tube three times to mix. If you do
not carefully mix, then the reaction may not proceed. Let the reaction proceed for
61
precisely 5.0 minutes (use a timer). Terminate the reaction by adding 300 L of 1.0 M
KOH and mix by inverting the tube three times or vortexing at high speed. KOH stops
the reaction by denaturing the enzyme. It also provides the alkaline solution required to
form the yellow p-nitrophenoxide ion. Adding KOH before adding 10 L of enzyme
fraction in tubes 5, 8, 11, and 14 prevents enzymatic activity and corrects for yellow
color that may appear due to nonenzymatic formation of p-nitrophenoxide. Add 300 L
of 1.0 M KOH to tubes 1-4 and mix by inverting the tube or vortexing.
Use the plastic cuvette and the spectrophotometer to read absorbances at 405 nm.
First set the absorbance of the UV-visible spectrophotometer to zero with the solution in
tube 1 (the blank) before reading the others. It is advisable to pour your samples back
into the appropriate microcentrifuge tube after you have read the absorbance, in case
you need to re-read them again later. In between samples, squirt distilled water from
your squeeze bottle into the cuvette, pour it into your waste container and then give it a
good shake to eliminate the remaining water. If you get negative values: check that
you are reading the absorbance at 405 nm, and make sure that you have blanked the
spectrophotometer with water (tube 1), and make sure that you are placing the cuvette
in the spectrophotometer in the same (and correct) orientation for each tube. If none of
these remedies work, contact your instructor or TA. Subtract the absorbance in tubes 5,
8, 11, and 14 from the average absorbances of the two following tubes. Compare these
corrected absorbances with the average from tubes 2, 3 and 4 (which contain a known
amount of p-nitrophenol) and calculate the moles p-nitrophenol formed per minute
during the enzyme-catalyzed reaction. Since 300 L of 5 x 10-5 M PNP contains 0.015
moles PNP, the average absorbance for tubes 2-4 can be used to calculate mol PNP
produced by the enzyme fractions using those corrected absorbances in the ratio:
Enzyme Activity Calculation
(for 10 L enzyme)
The moles PNP calculated in this way is for a 5 min reaction, therefore you must divide
the value by 5 to determine the enzyme activity in mol PNP / min (for 10 L
enzyme).
Calculating Total Protein (mg) per fraction:
The total protein in the fraction is calculated by multiplying the protein concentration that
you determined (mg/mL) by the total fraction volume that you recorded (mL). Report the
data in Table 4.
Calculating Total Units (mol PNP/min) of enzyme activity:
One unit of enzyme activity is the amount that catalyzes the formation of one mole pnitrophenol/min under the reaction conditions used in this experiment. From the enzyme
activity in each fraction, you can calculate the total number of units of enzyme in that
fraction. The total units in the crude homogenate, for example, is the activity (in moles
62
p-nitrophenol/min) times the total volume of the crude homogenate (e.g., 330 mL),
divided by the enzyme fraction volume (0.01 mL) used to determine activity. The total
units in other fractions are calculated in a similar way. Report the data in Table 4.
Calculating Specific Activity:
Specific activity is a measure of the purity of an enzyme preparation. It is the amount of
enzyme activity (molmin-1) per mg of protein. As the purity of an enzyme preparation
increases, its specific activity should also increase. To calculate specific activity for each
of the 4 enzyme fractions, divide the number of mol p-nitrophenol formed/min (from the
calculation above) by the mg protein providing that activity. To calculate mg protein
used from each fraction, multiply the protein concentration (in mg/mL) for the enzyme
fraction (determined by the Biuret test) by the volume of enzyme preparation used to
produce the activity measured (0.01 mL):
Amount of protein used
For Acid Phosphatase the units of specific activity are mol PNP / (min)(mg protein)
and the equation should be:
Specific Activity Calculation
Note: if you use the mol PNP / (min) that you calculated above, you do not need to
divide by 5 minutes again this is why there is a 1 in parenthesis in the equation).
Report the specific activity for each fraction in Table 4. In your conclusions section
comment on whether the purity of the preparation increased or decreased through the
several steps in isolating the enzyme.
Calculating purification factor:
The purification factor is calculated by dividing the specific activity of the fraction by the
specific activity of the crude homogenate. The larger this value is, the greater the purity
of the isolated protein.
In your results, report fraction volume, total protein per fraction, total units, specific
activity data, and purification factor in a protein purification table such as Table 4.
63
Table 4. Protein Purification Results.

Fraction Total
Total
Specific
purification
Volume Protein
Units
Activity
factor
(mL)
(mg) (mol PNP/min) (mol PNP/minmg)
crude homogenate
____
_____
__________
______________ _1.0_
55% acetone fraction
____
_____
__________
______________ _____
low% ammonium sulfate fraction
____
_____
__________
______________ _____
high% ammonium sulfate fraction
____
_____
__________
______________ _____
64
Grading guidelines: Isolation of Acid Phosphatase Formal Lab Report

Introduction (10 points)
Include the overall purpose of the experiment, as well as the basic principles of
the isolation techniques we used in this lab.
Discuss the methods used to analyze the protein concentration and the enzyme
activity.
Discuss the acid phosphatase enzyme and how to decide (when comparing
several fractions) which enzyme fraction is most pure.
Methods (2 points):
Cite the lab manual and note any changes you made
Data tables listing raw and corrected absorbance values should be included for
the biuret assay (Table 1), the PNP relationship (Table 2), and enzyme activity
assay (Table 3). Also, fill in Table 4.
Graphs of the standard curves for data in Tables 1 and 2, with r2 values above
0.95 and appropriate units should be included.
Sample calculations to be included (5 sample calculations total).
Protein concentration, enzyme activity (velocity), specific activity, total protein,
and total units of enzyme activity.
Always consider significant figures when reporting data.
Discuss the degree of success in isolating the enzyme, and the basis for your
comments. What data support your conclusion?
Discuss the sources of error and give a suggestion on how the process could be
improved for future classes.
Comment on the purity of the enzyme fractions. Did the purity increase or
decrease as you progressed through the isolation steps? Support your
conclusions with data from your results section.
Reference and Appendix (2 points):
Cite three references on enzyme isolations from the peer-reviewed literature.
Attach copies of your lab notebook pages. You should have quite a bit of
information in your lab notebooks for this lab!
65
Characterization of Acid Phosphatase

You have tested for acid phosphatase activity and know the general procedure for doing
this. It is now time for you to take your skills to the next level by designing and carrying
out two experiments that will characterize the enzyme you just isolated. Below are
several suggestions for experiments. However, if you would like to design another
experiment, you should discuss this possibility with your instructor. Conduct each
experiment individually, using the enzyme fraction that has the highest specific activity.
Your experimental design and proposed procedure must be written into your lab book
and approved by your instructor before you may begin. Each student will present their
results and conclusions to the class in an oral report using MS Powerpoint.
Available Reagents:
The cocktail from the Enzyme Isolation Lab containing (5 mM

p-nitrophenylphosphate (PNPP substrate), 0.1 M acetate at pH
5.2 (buffer) and 0.1% BSA (as an enzyme stabilizing agent).
50 mM PNPP (10x concentrated substrate)
1% BSA (an enzyme stabilizing agent)
Acetate buffer, 0.3 M, pH 5.2
Other Buffers, 0.3 M, at various pH: 4.0, 4.5, 5.0, 5.5, 6.0, 6.5,
7.0, 8.0 and 9.0
Inhibitors: 10 mM sodium fluoride and 10 mM sodium phosphate
1M KOH (for stopping the reaction)
5.0 x 10-5 M PNP (as the standard for product formation)
Available Equipment:
UV-Vis spectrophotometers,
Water Baths at C temperatures: 0-4 (on ice), 20 (room
temperature), 37, 50, 80, ~100 (boiling water bath).
Suggested Experiments
Kinetics
1. If you choose to determine Km and Vmax, you will make five cocktails containing
different substrate (p-nitrophenylphosphate) concentrations that range from 0.2
mM to 5 mM. Use MS Excel to graph the reciprocals of substrate concentration
and velocity (mol PNP/min) on the double reciprocal plot (Lineweaver-Burk).
Use the linear equation obtained from the double reciprocal plot to calculate Km
and Vmax. When deciding upon the substrate concentrations that you will use,
consider how they will look on the double reciprocal plot. Use at least three
concentrations below 1 mM and two at or above 1 mM.
66
2. To explore the effect of an inhibitor, use the substrate concentration range

listed above in the absence and presence of an inhibitor such as fluoride ion
(F-) or phosphate ion (PO43-). Stock solutions of sodium fluoride (10 mM) and
sodium phosphate (10 mM) will be available to you. Use a constant final
concentration of the inhibitor in the reaction mixture (in the range 1.0 to 3.0
mM) of sodium fluoride or sodium phosphate with varying substrate
concentrations. Use MS Excel to graph the reciprocals of substrate
concentration and velocity (mol PNP/min) in a Lineweaver-Burk plot to
determine the type of inhibition. Use the linear equation obtained from the
double reciprocal plot to calculate Ki and Vmax
Effect of pH
3. You can investigate the effect of pH on the reaction rate by making reaction
mixtures (cocktails) with different pH values. Everything else about the
reaction (e.g., time for reaction, temperature, substrate concentration) should
remain constant. Do the reaction over a range of pH values (at least five
different pH values) both above and below the pH where you expect maximum
activity. Plot velocity (mol PNP/min) versus pH.
Effect of Temperature
4. To determine the activation energy of the enzyme, Ea, run the enzyme
catalyzed reaction at different temperatures. Everything else about the reaction
(e.g., time for reaction, pH, substrate concentration, enzyme concentration)
should remain constant. In your experiment, use at least four temperatures in
the range of 0o to 50o C. Make sure you include the highest temperature, 50
o
C, as one of your choices. Plot the natural log (ln) velocity or rate of the
reaction (mol PNP/min) versus 1/T (in Kelvin), and from the slope calculate
the energy of activation, Ea, using the formula: slope = - (Ea/R), where R is the
gas constant, R = 8.314 J/mol K.
The activation energy, Ea, is calculated with the Arrhenius equation using the
rate constant, k, of the reaction: ln k = -(Ea/R)(1/T). In this study, you are
measuring the reaction rate of an enzyme catalyzed reaction which is
proportional to the rate constant and thus gives a parallel slope to the rate
constant data.
5. Another option is to test the effect of temperature on enzyme stability or
denaturation. Incubate different portions of the enzyme preparation for a fixed
time (at least 45 min) at five different temperatures in the range 1o to 100 oC.
Make sure you include the highest temperature, 100 oC, as one of your choices.
Then cool the samples and test each enzyme preparation for activity at 37o C
as you did in the Enzyme Isolation Lab. Plot velocity in mol PNP/min versus
incubation temperature in oC.
67
Instructions for the oral presentation

1. Talks should be about 10 minutes (no shorter than 8 min, no longer than 12 min).
2. Presentations are to be assembled and presented using Microsoft PowerPoint.
3. The final PowerPoint presentation is due and must be emailed to your instructor
no later than 11:00 AM on the day of your scheduled presentation.
4. Practice your talk out loud, preferably with an audience of your peers (they will give
you hints on how to improve).
5. This is a formal, professional talk and will be graded as such. The following rubric will
be used to grade the presentation.
Hint: Relax, everyone is on your side. Practice your talk out loud. Your presentation
does not need to be flashy; simple is often more elegant and effective.
CHEM 431Lab Biochemistry

Oral Presentation Assessment Rubric
Presenter: _________________________________________________
1. Description of the topic and scientific significance
____/5
Background. Why was the project undertaken? What is acid phosphatase?
Which enzyme fraction was chosen to characterize and why? Why is it important
to know these characteristics of an enzyme?
2. Discussion of the chemical nature of the topic
____/5
What was done? Which experiments were chosen? How were they conducted?
What is the chemical nature or biochemical basis of the experiments that were chosen?
3. Presentation and Analysis of Results
____/10
Data analysis and interpretation. Data tables showing original values.
Calculations are correct and show how results were derived.
Graphs are clear, showing trends in data and have correctly labeled axes.
Summary of what was learned. What do the results mean? What do they tell you
about the enzyme?
4. Visual aids, Verbal Comments, Professionalism.
Clarity and flow of information presented both verbally and visually.
Professionalism of presentation.
____/5
Total Points:
____/25
68
Appendix I: Introduction to UV-Visible Spectrophotometers

It is often necessary to accurately determine the concentration of a sample of protein,
nucleic acid, or other sample. The most frequently used instrument for determining the
concentration of samples is the UV-Visible spectrophotometer. The basic design of this
instrument is shown in the following diagram:
How a spectrophotometer works. Light is generated by a light bulb (1). This

light passes through a prism (2), separating the light into the different colors. The
filter (3) lets only the light with the correct wavelength (chosen by the operator)
through. This filtered light then passes through the sample (4) in the cuvette. The
detector (5) determines the amount of light that passed through the sample. The
amplifier (6) then increases the signal so that the output will be given in easy-tounderstand units by the output display.
For solutions, the amount of coloration is proportional to the concentration of the

compound absorbing the wavelength of light corresponding to that color; this
phenomenon is described by Beers law (or Beer-Lamberts law). This is very useful
because it allows us to plot the absorbance of known concentrations of a given solution
vs their absorbance at a particular wavelength of light. The Beer-Lambert equation is:
A=xlxc
Where A = Absorbance (a unit-less value)
= molar extension coefficient (Abs units / M of samplepathlength in cm)
l = the path length in cm (usually = 1)
c = the molar concentration of the sample
It is important to note that UV-Vis spectrophotometers are often used for a second type
of measurement called optical density. Optical density is very different than absorbance,
69
as this value reflects the ability of the sample to deflect light so that it does not pass to
the detector, rather than to actually absorb light at a specific wavelength. This is
frequently used for estimating the concentration of cells (most commonly for bacteria
such as E.coli).
UV-Visible spectrophotometers have detection limitations that may not be obvious to the
operator. High end UV-Visible spectrophotometers can read and report absorbance
values (and optical density (O.D.) from 0.0001 up to around 5.0. You will be using a
standard quality instrument that can read absorbance values between 0.001 and
approximately 2.0. Keep this limitation in mind while you are working in the
laboratory and reporting your data.
The basis of this limitation is revealed in the mathematical calculation of absorbance.
Absorbance, A, of a solution is calculated as the log of the ratio of the incident light
going into the solution divided by emitted light coming out of the solution at a given
wavelength:
A = log10 Io/I
where Io is the incident light, and I is the emitted light.
Thus, an absorbance of 3 indicates, for example, 1000 photon input with only 1photon
output. This means 99.9% of the light is absorbed by the solution. A very high quality
spectrophotometer is needed to measure such a small photon output.
70
Instructions for UV-Visible Spectrophotometers (Beckman DU-520)

Turn on the instrument with the toggle switch on the rear panel (left rear). Allow the instrument
to warm up for 30 minutes before using. Note that if you are reading absorbance values in the
UV range, that you must use a quartz cuvette, while you can use a disposable plastic cuvette if
you are reading in the visible range. Note that these machines do not have touch screen
inputs; use the buttons below the menu items on the screen.
For Fixed Wavelength () Measurements (Fixed )
In the Main Menu Screen
Select Fixed
For absorbance measurements be sure that the View option reads:
View: Abs, then
Select OPTIONS. Select GO TO Select 1.
Enter the wavelength desired using the number pad (e.g., 280 or 540 nm).
Press ENTER to select the entered wavelength.
Press EXIT. Press EXIT again.
Now place the cuvette with the blank solution in the cuvette holder and press BLANK to
zero the instrument.
Take the blank cuvette out and put the cuvette with your sample in and press READ.
The absorbance value will appear on the screen. Write the absorbance value in
your lab notebook.
For UV-Visible Wavelength Scans ( scans):
In the Main Menu Screen
Select Scan. Select OPTIONS.
Select min. Input the minimum wavelength of the scan, e.g., 240, using the
number pad. Press ENTER.
Select max. Input the maximum wavelength of the scan, e.g., 320, using the
number pad. Press ENTER.
Press EXIT.
Now place the cuvette with the blank solution in the cuvette holder and press
BASELINE.
Take the blank cuvette out and put the cuvette with your sample in and press START
SCAN.
Once the completed UV-Vis scan appears on the screen, press CURSOR and then
press the CURSOR toggles (either the left or right arrow) to move the vertical line
and find the absorbance at 260 nm and 280 nm. The absorbance value at the
given wavelength appears below the screen (see figure below).
Make sure that the dial on the splitter box is positioned so that it points to your
Spectrophotometer (the letter of the machine is on it).
Press the PRINT key on the key pad. Press the Print Graph option. Include it in your
lab report.
71
APPENDIX II
Linear Regression Analysis: Excel 2007 Methods and Instructions
Note: These instructions are written for experiment #2, but can be applied to
others with small changes.
1.
Input your data (from the known solutions only!) into

Excel, as is shown in the example on the right.
Important note: Do not put units into the numerical
cells or Excel will be unable to plot the data.
2.
Select the cells that contain your data by

highlighting them with the mouse.
3.
Select Insert from the menu at the top of the screen, and then click on the box
listed as Scatter (use the scatter with only markers option located on the upper
left of the options shown) within the Charts menu. A plot will appear on your
screen.
4.
Click on this
button
(scatter).
Then select
the option
that appears
in the upperleft hand
corner. A
chart will
appear near your data.
5.
Some options will appear at the

top of the page: press the down
button (red arrow) twice and
then select the option shown on
the right (red box). The resulting
plot is shown on the next page.
Continue to the next page.
Concentration (mg/dL)
0
50
100
150
200
250
ABS
0
0.110
0.240
0.370
0.480
0.580
72
6.
Click on the figure legend and press backspace to delete.
7.
Click on the title. Change the title to Glucose Concentration vs AbsXXX, where
the XXX is the wavelength at which you made your absorbance recordings.
8.
Click on the X-axis title and change this to Glucose Concentration (mg/dL)
9.
Click on the Y-axis title and change this to Abs at XXXnm. The XXX should be
your wavelength. Your plot should now look like this:
Glucose Concentration vs Absxxx

0.7
y = 0.0024x + 0.001
R = 0.998
Abs at XXXnm
0.6
0.5
0.4
0.3
0.2
0.1
0
0
50
100
150
200
Glucose Concentration (mg/dL)
Continue to the next page.
250
300
73
Notes:
The R2 value gives an indication of how precisely the line fits the data (1.0 =
perfect correlation, 0 = no correlation). The linear equation is a mathematical
relationship between your input variable (x) and the output variable (y) and can
be used to determine the concentration of your unknown sample.
Using the Equation

First, you need to take the average of the absorbances for each of your unknown
samples. Lets say that the average absorbance for your unknown is 0.4256 (dont
worry about significant figures until later). This number is plugged into the: y = 0.0024x
+ 0.001 equation (from the plot) into the y position, giving:
0.4256 = 0.0024x + 0.001
Next, solve for x:
(0.4256 0.001) / 0.0024 = x
X = 176.9166 mg/dL
If your absorbance readings only had three significant digits, then this would be rounded
to 177 mg/dL. Ask yourself, Does this answer make sense? In this case it does, since
our absorbances fall between the values obtained for 150 and 200 mg/dL, and our
calculation gives us a result within this range.
74
Appendix III: Analyzing Agarose Gel Electrophoresis of DNA

There are many pieces of information that can be gleaned from the bands in an agarose
gel. The most obvious examples of this are the size of the bands of DNA and the
quantity of DNA in the bands. This brief tutorial will discuss how to determine the size
(length in bp) and concentrations of bands of DNA in agarose gel, along with showing
examples of other phenomena frequently encountered when performing agarose gel
electrophoresis of DNA samples.
Estimating the size of DNA fragments on a agarose gel
It is not normally necessary to determine the precise size of DNA band on an agarose
gel, as estimation by comparison to the ladder is usually sufficient. However, there are
times when it is necessary to determine the size of a nucleic acid (this can also be used
to determine the size of RNA molecules). Although it is possible to rely on software for
this task, it is frequently more laborious than physically measuring and calculating the
size of the bands by hand. Performing this task by hand is a simple two-step process:
(1) measuring the distance that bands in the DNA ladder (marker) and the unknown
DNA band have migrated through the gel, and (2) creating a standard plot for the bands
in the ladder so that the size of unknown band can be determined.
Figure 1. How to measure the migration

distances for bands on an agarose gel.
Continue to next page
75
Table 1. (Ladder DNA) Data collected from

the gel in Figure 1. The first column for the
ladder DNA represents the measured
distances (in mm) that the given bands
migrated. The known size of the bands (in
base pairs) is shown in the middle column.
The right column is the log of the values in the
middle column. Columns one and three were selected to make the graph shown in
Figure 2. (Unknowns) The size of the unknown bands was determined by measuring
the migration distance (third column) and taking the log of these values (second
column). These numbers were then plugged into the equation provided by the plot
shown in Figure 2 to generate the log of size of the bands in base pairs (shown in red).
Note: The numbers of base pair are rounded off to the nearest base pair, as you
obviously cannot have fraction of a base pair.
Figure 2. Plot of the log of the migration
distance of the bands vs the size of the
bands in the ladder. Measurements
were made from Figure 1 and the data
comes from Table 1. The equation: y = 0.0096x + 4.1826 was used to calculate
the size (in log bp) of the unknown
bands, using the migration distances.
x
The antilog (10 ) of the log (bp) values, shown in red in Table 1, were taken to convert
these number into the base pair values shown in red in Table 1.

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CHEM 431 LAB MANUAL

The Format of Formal Lab Reports ...

Automatic Pipettes: Instruction for Use .

Lab #1: Checking the Accuracy and Precision of Pipettes ...

Lab #2: Quantitative Measurement of Proteins ....

Lab #3: Electrophoresis of Serum Proteins Using SDS-PAGE ....

Drying Polyacrylamide Gels in Cellophane.

Analyzing the gel using the Image J Software ..

Lab #4: Restriction Enzyme Analysis of Circular DNA ...

Lab #5: Isolation of an Enzyme: Acid Phosphatase

Lab #6: Characterization of Acid Phosphatase ....

Instructions for the oral presentation ..

Appendix I: Introduction to UV-Vis Spectrophotometers ..

Instructions for UV-visible Spectrophotometers (Beckman DU-520) ..

Appendix II: Linear Regression Analysis: Excel 2007

Appendix III: Analyzing Agarose Gel Electrophoresis of DNA .....

General Lab Rules

Pipette tip holder

Test tube brush

Test tube clamp

Test tube rack

Microfuge tube rack

Automatic Pipettes: Instructions for Use

called the p1000 pipette

called the p200 pipette

called the p20 pipette

Reading the volume set-point window

METHOD FOR USE

ADDITIONAL OPERATING HINTS

Checking the Accuracy and Precision of Pipettes

Equipment and Reagents Needed

A clean 50 mL beaker containing 40 mL of distilled water (white tap)

An example Excel spreadsheet is posted to the course blackboard

Data Collection Sheet

Quantitative Measurement of Proteins

absorbance at the given wavelength

Figure 1. An absorption spectrum of a given substance over a

Bradford Method to Measure Protein Concentration

Figure 2. Coomassie Brilliant Blue G-250

Equipment and Reagents Needed

The p1000, p200, and p20 micropipettes

Stock BSA volume

[BSA] after dilution

Transfer by pouring 3.0 mL of Bradford reagent (it is located in the refrigerator

Take your samples, a water bottle, a waste container, and a disposable

Ultraviolet Absorption to Measure Protein Concentration

Equipment and Reagents Needed

The p1000, p200, and p20 micropipettes

Take the following items to an available UV-Vis spectrophotometer: your

Plot the absorbance at 280 nm versus concentration for the samples of

Biuret Method to Measure Protein Concentration

Figure 1. Protein-copper complex.

Equipment and Reagents Needed

The p1000, p200, and p20 micropipettes

Dilute the 1% solution of BSA to prepare 0.600 mL (= 600 L) samples

Plot the absorbance at 540 nm versus concentration for the samples of

Electrophoresis of Serum Proteins

peptide bond formation is shown in the figure on the right, where a

Returning to electrophoresis, each of these factors can determine

standard (nondenaturing) conditions. From

Figure 1. Schematic of the mobility of

Read this step