Structure of Agarose

Polyacrylamide gel

Electrophoretic mobility in the gel

logE = logE’- KrG

•E- electrophoretic mobility•E’- mobility in sucrose solution•Kr- retardation coefficient•G- Gel concentration

Solubilizers

• Urea- conc. 3-12M, Disrupt Hydrogen bonds

• SDS- Anionic detergent, imparts negative charge, Disturbs hydrophobic interactions.

• CTAB- Cationic detergent.

• B- mercaptoethanol- Distrubs disulphide linkage

Electrophoretic Procedure

Models of Electrophoresis

Agarose gel electrophoresis

• Structure of Agarose gel• Preparation of gel• Submerged gel electrophoresis• Detection• Fluorescent method for NA.• Staining for proteins

Structure of Agarose Gel

PAGE

• Structure of gel

• Components of gel

• Electrophoretic run

• Detection

Discontinuous PAGE

Applications

• Separation of proteins• DNA Sequencing• Western Blotting• Determination of molecular weight of proteins

Isoelectric Focussing

• Introduction• Principle• Establishing the pH gradient- carrier

ampholytes• Stabilization against Convection• Procedure• Separation of protein from carrier ampholytes• Applications

Determination of isoelectric Point of a protein

• Measure the mobility of the protein at several pH values.

• Plot mobility values against the pH values.

• Plot intercept at zero mobility.

Establishing the pH gradient- carrier ampholytes

• Isomers and homologs of aliphatic polyamino polycarboxylic acids.

• R- N-(CH2)n- N - (CH2)n- COOH

Where R- (CH2)n- COOH, H• n- less than 5

• Ex.- ampholine, Pharmalyte, Bio-lyte.

• Used in 1% concentration

Properties

• Carrier ampholytes must dictate the pH course, should have a certain buffering capacity at their isoelectric point.

• Should have a conductance at their isoelectric point.• Low molecular weight.• Should be soluble in water.

• Should have law absorption at 280nm.

Stabilization against convection

1)Density Gradient- • Uncharged solutes dissolvable in water• Should not react with proteins, low metal content, high purity• Ex.- sucrose- 50% , up to pH 10.protective action on protein.• Glycerol, ficoll, sorbitol, ethylene glycol, dextran

2)Gel- as anticonvectant, 7.5% acrylamide• High molecular weight protein- .5%

3) Zone convection

Procedure- Column- density gradient Plate- polyacrylamide gel

Separation of protein from carrier ampholytes

• Avg. mol.wt of ampholyte- 800D, • Avg. mol.wt of protein- 10000D

• Method of separation –

1) Dialysis – 99% efficient, Slow process

2) Gel filtration – Sephadex G-50

3) Ammonium sulphate precipitation

4)Ion exchange chromatography

5) Partition chromatography

Applications

• Separation and identification of serum proteins.

• Used in food and agricultural industries.

• Forensic & human genetics labs

• Research in enzymology, immunology & membrane biochemistry

Two dimensional electrophoresis

• Combination of isoelectric focussing & SDS-PAGE.

• Can resolve 5000 proteins in individual bands.

• Uses isoelectric pH and molecular weight combination