This document discusses various electrophoretic techniques used to separate biomolecules like proteins and DNA. It describes the principles and procedures of agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), discontinuous PAGE, isoelectric focusing, and two-dimensional electrophoresis. These techniques separate molecules based on properties like their electrophoretic mobility, molecular weight, and isoelectric point within different types of gels and buffers under an applied electric field. The document also outlines solubilizers, detection methods, and applications of these electrophoretic procedures.

1. Structure of Agarose

3. Polyacrylamide gel

4. Electrophoretic mobility in the gel
logE = logE’- KrG
•E- electrophoretic mobility
•E’- mobility in sucrose solution
•Kr- retardation coefficient
•G- Gel concentration

5. 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

6. Electrophoretic Procedure

7. Models of Electrophoresis

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

12. Structure of Agarose Gel

14. PAGE
• Structure of gel
• Components of gel
• Electrophoretic run
• Detection

17. Discontinuous PAGE

22. Applications
• Separation of proteins
• DNA Sequencing
• Western Blotting
• Determination of molecular weight of proteins

24. Isoelectric Focussing
• Introduction
• Principle
• Establishing the pH gradient- carrier
ampholytes
• Stabilization against Convection
• Procedure
• Separation of protein from carrier ampholytes
• Applications

27. 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.

28. 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

29. 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.

30. 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%

31. 3) Zone convection

32. Procedure- Column- density gradient
Plate- polyacrylamide gel

33. 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

34. Applications
• Separation and identification of serum proteins.
• Used in food and agricultural industries.
• Forensic & human genetics labs
• Research in enzymology, immunology & membrane
biochemistry

35. Two dimensional electrophoresis
• Combination of isoelectric focussing & SDS-PAGE.
• Can resolve 5000 proteins in individual bands.
• Uses isoelectric pH and molecular weight
combination