Ultrafiltration

Based on a pressure differential across the semipermeable membrane to drive permeable materials through the membrane Separation of molecular size, shape and/or charge

Membranes are chosen based on: on:

Flow rate Molecular specificity MWCO

Composed of two layers: layers:

Thin, semipermeable surface made from variety of materials Thick, inert support base

Application
   

Desalting buffers or other solutions Clarification of turbid solutions Collection of precipitates for analysis Harvesting of bacterial cells from fermentation broths Concentration of biomolecule solutions

Gel Filtration Chromatography



Separation of macromolecules in mixture based on differences in molecular size and shape Column is packed with porous beads that act as molecular sieve

 

Mobile phase - eluent Stationary phase

Beads composed of spongesponge-like matrices of varying pore size

Characteristics of Gel Filtration Media 1. Chemical inertness 2. Low content of ionic groups 3. Uniform pore size and particle size 4. High mechanical rigidity

Dextran high molecular mass of glucose produced by Leuconostoc mesenteroides Agarose a linear of polymer of alternating DDgalactose and 3,63,6anhydroanhydro-L-galactose from red algae


Polyacrylamide made from the polymerization of acrylamide and N,N methylenebisacrylamide

Fractionation Range (kD)

Sephadex G-10 Sephadex G-25 Sephadex G-50 Sephadex G-100 Sephadex G-200 Bio-Gel P-2 Bio-Gel P-6 Bio-Gel P-10 Bio-Gel P-30 Bio-Gel P-100 Bio-Gel P-300 Sepharose 6B Sepharose 4B Sepharose 2B 0.05-0.7 1-5 1-30 4-150 5-600 0.1-1.8 1-6 1.5-20 2.4-40 5-100 60-400 10-4,000 60-20,000 70-40,000

Sample Problem
What is the order of elution of the following proteins from a Sephadex G-50 column: Proteins Molecular Mass (kD)
Catalase -Chymotrypsin Concanavalin B Lipase Myoglobin 222 21.6 42.5 6.7 16.9

Exclusion Limit molecular mass of the smallest molecule unable to penetrate the pores of a given gel. Vt = Vx + V0

Where, Vt the total bed volume of the column Vx volume occupied by the gel beads V0 volume of the solvent space surrounding the beads

Ve

volume of the solvent required to elute the solute from the column after it has first contacted from the gel.

Sample Problem A gel-chromatography column of Bio-Gel P-30 gelBioPwith a bed volume of 100 mL is poured. The elution volume of the hexokinase (96 kD) on this column is 34 mL. That of an unknown protein is 50 mL. What is the void volume occupied by the gel and the volume occupied by the gel?

SetSet-up
   

Column
determination of bed volume

Bed Support Polyethylene tube Media

determine the amount of water/buffer needed to swell the gel Hydration time

Reservoir

Chromatography proper
1. 2. 3. 4.

Determine Vo using Blue Dextran Application of standard Application of sample Determination of MW

Estimation of MW
Relative Elution Volume = Ve / V0

Applications


 

Separating substances with different molecular weights Desalting protein solutions Determining the molecular weight or size of native or denatured globular proteins under variety of conditions

Based on Charge Electrophoresis



Charged molecules moved in an electric field thru a support medium. Used for separation for separation of proteins and nucleic acids Positively charged molecules cathode Negatively charged molecules anode

 

Support medium: paper, cellulose acetate, starch, agarose, polyacrylamide Rate of migration not only depends on charge but also size and shape.

Paper Electrophoresis
Steps:
Application Development Visualization

Sample Problem
A mixture of amino acids consisting of arginine, cysteine, glutamic acid, histidine, leucine and serine is applied using a buffer at pH 7.5. What are the direction of these amino acids?

Isoelectric focusing


Involves electrophoresis of protein mixtures thru stable pH gradient medium. Separation based on the isoelectric points of proteins

Protein will migrate to the region where pH = IpH. Proteins are focused into a narrow band. Separation can be thin as 0.01 pH unit

How to make a stable pH gradient?

The pH gradient is established by the use of ampholytes. These are low molecular weight amphoteric molecules which will migrate in an electric current to their pI, thus producing a pH gradient.

Sample Problem
What would be the relative arrangement of the following proteins subjected to isoelectric focusing: IpH Insulin 5.4 Cytochrome c 10.6 Histone 10.8 Myoglobin 7.0 Ribonuclease A 7.8

Gel Electrophoresis


Support medium:
Gel is matrix of polymers forming subsubmicroscopic pores Use of either agarose or polyacrylamide gel

Agarose Gel Electrophoresis (AGE)



Agarose is a linear polysaccharide (average molecular mass about 12,000) made up of the basic repeat unit agarobiose, which agarobiose, comprises alternating units of galactose and 3,63,6-anhydrogalactose.

 

Dissolved in buffer solutions, melted by heating and poured Differences in ionic strength of buffer solutions provide differences in rate of migration of solute. Electrophoresis buffer 1. Tris-Acetate-EDTA Tris-Acetate(TAE) 2. Tris-Borate-EDTA Tris-Borate(TBE)

Agarose gel have large range of separation but relatively low resolving power.
Efficient Range of Separation of linear DNA Molecules (kb) 60-5 20-1 10-0.8 7-0.5 6-0.4 4-0.2 3-0.1

Amount of Agarose in Gel (%) 0.3 0.6 0.7 0.9 1.2 1.5 2.0

Sample is loaded on the wells of the gel. Loading dye is a negatively charged indicator added with the sample because: it contains sucrose or glycerol which increases the density of the DNA sample so that it sinks to the bottom of the well, and; it contains dyes that can indicate how far the DNA samples have run during electrophoresis.

Gel acts as molecular sieves. sieves.

Small molecules Large molecules

migrates faster migrates slower

Factors affecting migration: 1. concentration of agarose 2. conformation of DNA

Visualization  Ethidium bromide

It is a fluorescent dye that intercalates between the bases of nucleic acids.

Polyacrylamide Gel Electrophoresis (PAGE)



Polyacrylamide are formed by the polymerization of acrylamide in aqueous solution in the presence of small amounts of a bifunctional crosslinker.

Catalysts for the Polymerization of Acrylamide and Crosslinker



Ammonium persulfate Used in the free radical catalytic polymerization of acrylamide and bisbisacrylamide. TEMED is used to catalyse to decomposition of the persulfate ion to form a sulphate ion and a free radical form of the sulfate ion: eS2O8-2 + e- SO4-2 + SO4-2 N,N,N',N'N,N,N',N'-Tetramethylethylenediamine (TEMED) catalyses the decomposition of the persulphate ion to give a free radical.

Polyacrylamide have high resolution power but a relatively low range of separation.

T represents the total percentage concentration (w/v) of monomer (acrylamide plus crosslinker) in the gel. C refers to the percentage of the total monomer represented by the crosslinker. Example: An 8%, 19:1 (acrylamide/bisacrylamide) gel would have a T value of 8% and a C value of 5%.

Tracking Dye An dye added to the sample buffer to indicate the progress of the electrophoresis run. Because the dye is charged it will move under the influence of the electrophoretic field.

Ex. Bromophenol blue This dye has a negative charge in the buffer. The dye is also small and thus moves faster than the proteins in the sample.

Visualization
1.

Staining
a. Use of Coomasie brilliant blue dye

b. Use of fluorescamine

3. Immunoblot / Western blot

4. Autoradiograph

If sample is radioactive, a sheet X-ray film is Xexposed over gels for a period of time, then developed. The level of radioactivity can be measured by scintillation counter.

PAGE vs AGE
1.

2.

3.

An agarose gel, the pore size is large, so molecular sieving, i.e. separation by size, will not occur for smaller DNA fragments and most proteins. Additionally, by altering the total concentration of monomer in the gel and the ratio of acrylamide to bis, the pore size with a polyacrylamide gel can be altered in a reproducible manner. Acrylamide and Bis are synthetic chemicals; there are virtually no batch to batch differences.

SDSSDS-PAGE


Sodium Dodecyl Sulfate (SDS) This is a detergent because it contains a hydrophobic region, attached to a hydrophilic group making it amphipathic. It is a very amphipathic. strong detergent which denatures proteins by binding to the polypeptide backbone.

The end result has two important features: 1. All proteins retain only their primary structure and 2. All proteins have a large negative charge which means they will all migrate towards the positive pole when placed in an electric field.

SDSSDS-PAGE can be use to determine the number of subunits in a protein and MW of each of the subunit. Use 2-mercaptoethanol to cleave disulfide linkage 2-

The Rf is calculated as the ratio of the distance migrated by the molecule to that migrated by a marker dye-front.

Types of Buffer Systems in Electrophoresis

1.

Continuous system It has only a single separating gel and uses the same buffer in the tanks and the gel.

2. Discontinuous System
a. Stacking gel - a non-restrictive large pore nongel (containing only 4% acrylamide)
It is designed to sweep up proteins in a sample between two moving boundaries so that they are stacked into micrometer thin layers when they reach the separating gel.

b. Resolving gel It is responsible for actually separating polypeptides by size.

Two Dimensional Gel Chromatography

IonIon-Exchange Chromatography


Separation of molecules mainly based on charge It is reversible exchange of ions in solution with ions electrostatically bound to an adsorbent.

Ion Exchanger
Inert support medium to which positive or negative functional groups are covalently bound.

Anion Exchanger

Cation Exchanger

Anion Exchanger
Cellulose - DEAE Cellulose - QAE

Cation Exchanger
Cellulose CM

The composition of gradient falls into two categories: 1. gradient of increasing ionic strength 2. gradient of changing pH

Sample Problem
In what order will the following proteins be eluted from a CM-cellulose ion exchange CMcolumn by an increasing salt gradient at pH 7: IpH Fibrinogen 5.8 Hemoglobin 7.1 Lysozyme 11.0 Pepsin 1.0 Ribonuclease A 7.8