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1. 2. 3. 4. 5. 6. Definitions Theory of Electrophoresis Electrophoretic Technique General Procedures Types of Electrophoresis Technical Considerations
1. DEFINITIONS
Electrophoresis Migration of charged solutes in a liquid medium under an electrical field
Many biological molecules have ionisable groups eg. amino acids, proteins, nucleotides, nucleic acids Under an electric field -> charged particles migrate to anode (+) or cathode (-)
Zone Electrophoresis
Migration of charged molecules Support medium porous eg. CA or agarose can be dried & kept
Isotachophoresis Migration of small ions Discontinuous electrolyte system leading electrolyte (L- ions, high mobility) & trailing electrolyte (T- ions)
3. ELECTROPHORETIC TECHNIQUE
3a. Instrumentation & Reagents
3c. Buffers
To carry applied current & to fix the pH => determine electrical charge & extent of ionization => which electrode to migrate Ionic strength of buffer thickness of ionic cloud -> migration rate -> sharpness of electrophoretic zones [ion] -> ionic cloud -> movement of molecules Barbital buffers & Tris-boric acid-EDTA buffers
4. GENERAL PROCEDURES
4a. Separation Place support material in EP chamber Blot excess buffer from support material Place support in contact with buffer in electrode chamber Apply sample to support
cont. Separation
Separate component using constant voltage or constant current for length of time Remove support, then -> dry or place in fixative -> treat with dye-fixative -> wash excess dye -> dry (agarose) or put in clearing agent (CA membs)
By densitometry electrophoretic strip moved past an optical system absorbance of each fraction displayed on recorder chart
e. Two-dimensional Electrophoresis
Cont. AGE
Advantages: low affinity for proteins shows clear fractions after drying low melting temp -> reliquify at 65oC Disadvantage: poor elasticity -> not for gel rod system -> horizontal slab gels
cont. CAE
Method: wet CA in EP buffer load sample about 1/3 way along strip stretch CA in strips across a bridge place bridge in EP chamber -> strips dip directly into buffer after EP, stain, destain, visualise proteins For diagnosis of diseases change in serum protein profile
5c. Polyacrylamide Gel Electrophoresis (PAGE) Tubular-shaped EP cell -> pour small-pore separation gel -> large-pore spacer gel cast on top -> large-pore monomer solution + ~3ul sample on top of spacer gel Electrophoresis -> all protein ions migrate thru large-pore gels -> concentrate on separation gel -> separation due to retardation of some proteins
Average pore size in 7.7% PAGE separation gel about 5nm -> allow most serum proteins to migrate -> impedes migration of large proteins eg fibrinogen, 1-lipoprotein, 2macroglobulin
Advantages: thermostable, transparent, strong, chemically inert wide range of pore sizes uncharged -> no endosmosis Disadvantages: carcinogenic
Denaturing PAGE/SDS-PAGE
What is SDS-PAGE?
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis A procedure to separate proteins and determine their Molecular Weights.
Boil sample for 5 mins in sample buffer containing -mercaptoethanol & SDS -mercaptoethanol: reduce disulfide bridges SDS: binds strongly to & denatures proteins Proteins denatured -> opens into rodshaped structures -> separate based on size Use: To assess purity of protein To determine MW of protein
apply a potential difference across gel anode -> area with lowest pH cathode -> area with highest pH proteins migrate until it arrives at pH = pI wash with fixing solution to remove ampholytes stain, destain, visualise Separations of proteins with 0.01 to 0.02pH unit differences (Fig 7-4)
Medium pH range
(pH 4-7)
pI 4 kDa 116 97 81 66 55 45 5 6 7
30
21 14
(5.0-6.0)
MW (kDa)
30
21
14
Immunoblotting
Northern blot (RNA)
IMMUNOASSAYS
1. Basic Concepts & Definitions 2. Measurement of Antibody Affinity 3. Quantitative Methods competitive & noncompetitive assays
1. BASIC CONCEPTS & DEFINITIONS Immunoassay: use of antibodies to detect analyte 1a. Antibodies Immunoglobulins that bind to Antigens 5 classes: IgG, IgA, IgM, IgD, IgE 1b. Immunogen Protein or a substance coupled to a carrier When introduced into foreign host -> induce Ab to form 1c. Antigen Any material which can react with Ab May not induce Ab formation
1d. Antigen-Antibody Binding Ab molecules have specific binding sites -> bind tightly to Ag -> cause pptn/neutralization/ death Binding of Ag to Ab due to van der Waals forces hydrophobic interactions charged group attractions
r/c = nK rK
r = no. of molecules of bound Ag per Ab molecule c = concn of free Ag n = valency of Ab
Plot r/c vs r => Scatchard Plot Straight line with slope k x intercept gives n y intercept gives nK
K (liters/mole) measures affinity of complex
3. QUANTITATIVE METHODS
Read & Understand from Tietz Fundamentals: Radial Immunidiffusion Immunoassay Electroimmunoassay Turbidimetric & Nephelometric Assays
Labels Ag (Ag*) & unlabeled Ag compete for binding to Ab The probability of Ab binding to Ag* is inversely to [Ag]
(ii) Sequential Competitive Assay Step 1: unlabeled Ag mixed with excess Ab -> binding allowed to reach equilibrium Step 2: Ag* added sequentially -> equilibrate After separation -> det bound Ag* -> calculate [Ag] Larger fraction of Ag bound to Ab than in simultaneous assay If k1 >> k2 -> in Ab:Ag -> in Ag* binding Provide two- to four- fold improvement in detection limit
b. Noncompetitive Immunoassays
i. Used when have excess reagent
Immobilization of Ab to support Passively adsorption or bind covalently Direct or indirect attachment (Table 9-3) ii. Ag allowed to react with Ab -> wash other proteins iii. Add labeled Ab (conjugate) -> reacts with bound Ag iv. Determine bound label -> [Ag*] or its activity is [Ag]