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Structure of Agarose
Polyacrylamide gel
Electrophoretic mobility in the gel 
logE = logE’- KrG 
•E- electrophoretic mobility 
•E’- mobility in sucrose solution 
•...
Solubilizers 
• Urea- conc. 3-12M, Disrupt Hydrogen bonds 
• SDS- Anionic detergent, imparts negative charge, Disturbs 
hy...
Electrophoretic Procedure
Models of Electrophoresis
Agarose gel electrophoresis 
• Structure of Agarose gel 
• Preparation of gel 
• Submerged gel electrophoresis 
• Detectio...
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 prote...
Isoelectric Focussing 
• Introduction 
• Principle 
• Establishing the pH gradient- carrier 
ampholytes 
• Stabilization a...
Determination of isoelectric Point of a protein 
• Measure the mobility of the protein at several 
pH values. 
• Plot mobi...
Establishing the pH gradient- carrier ampholytes 
• Isomers and homologs of aliphatic polyamino polycarboxylic 
acids. 
• ...
Properties 
• Carrier ampholytes must dictate the pH course, 
should have a certain buffering capacity at their 
isoelectr...
Stabilization against convection 
1)Density Gradient- 
• Uncharged solutes dissolvable in water 
• Should not react with p...
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 
• Meth...
Applications 
• Separation and identification of serum proteins. 
• Used in food and agricultural industries. 
• Forensic ...
Two dimensional electrophoresis 
• Combination of isoelectric focussing & SDS-PAGE. 
• Can resolve 5000 proteins in indivi...
Electrophoresis new1
Electrophoresis new1
Electrophoresis new1
Electrophoresis new1
Electrophoresis new1
Electrophoresis new1
Electrophoresis new1
Electrophoresis new1
Electrophoresis new1
Electrophoresis new1
Electrophoresis new1
Electrophoresis new1
Electrophoresis new1
Electrophoresis new1
Electrophoresis new1
Electrophoresis new1
Electrophoresis new1
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Electrophoresis new1

  1. 1. Structure of Agarose
  2. 2. Polyacrylamide gel
  3. 3. Electrophoretic mobility in the gel logE = logE’- KrG •E- electrophoretic mobility •E’- mobility in sucrose solution •Kr- retardation coefficient •G- Gel concentration
  4. 4. 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
  5. 5. Electrophoretic Procedure
  6. 6. Models of Electrophoresis
  7. 7. Agarose gel electrophoresis • Structure of Agarose gel • Preparation of gel • Submerged gel electrophoresis • Detection • Fluorescent method for NA. • Staining for proteins
  8. 8. Structure of Agarose Gel
  9. 9. PAGE • Structure of gel • Components of gel • Electrophoretic run • Detection
  10. 10. Discontinuous PAGE
  11. 11. Applications • Separation of proteins • DNA Sequencing • Western Blotting • Determination of molecular weight of proteins
  12. 12. Isoelectric Focussing • Introduction • Principle • Establishing the pH gradient- carrier ampholytes • Stabilization against Convection • Procedure • Separation of protein from carrier ampholytes • Applications
  13. 13. 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.
  14. 14. 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
  15. 15. 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.
  16. 16. 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%
  17. 17. 3) Zone convection
  18. 18. Procedure- Column- density gradient Plate- polyacrylamide gel
  19. 19. 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
  20. 20. Applications • Separation and identification of serum proteins. • Used in food and agricultural industries. • Forensic & human genetics labs • Research in enzymology, immunology & membrane biochemistry
  21. 21. Two dimensional electrophoresis • Combination of isoelectric focussing & SDS-PAGE. • Can resolve 5000 proteins in individual bands. • Uses isoelectric pH and molecular weight combination

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