Electrophoresis

brief

1. ELECTROPHORESIS BY Pintu Choudhary Master of Technology ( Food Science and Technology) Indian Institute of Crop Processing Technology Ministry of Food Processing Industries Government of India Thanjavur - 613005

2. Electrophoresis Electrophoresis is a method whereby charged molecules in solution, chiefly proteins and nucleic acids, migrate in response to an electrical field . As an analytical tool, electrophoresis is simple, rapid and highly sensitive. It is used for analysis and purification of very large molecules (proteins, nucleic acids) for analysis of simpler charged molecules (sugars, amino acids, peptides, nucleotides, and simpler ions).

3. Basic Principle It is the process of moving charged bio molecules in solution by applying an electrical field across the mixture. Bio molecules moved with a speed dependent on their charge, shape, and size and separation occures on the basis of molecular size.

4. Principle When charged molecules are placed in an electric field, they migrate toward either the positive (anode) or negative (cathode) pole according to their charge.

5. Factors Affecting Electrophoretic Mobility Number of parameter of electrophoretic system: For zone EP, type of support medium chosen, and if it is a gel, its pore size Ph. of the electrophoresis buffer Ionic composition of buffer Applied voltage Temperature

6. Supporting Medium Selection is based on following consideration Size of the molecules to be analyzed Quantity of sample available Cost of support medium to be used Availability of suitable equipment Purpose of the analysis Time to run the analysis Expertise of the operator

7. Types of Electrophoresis Affinity electrophoresis Capillary electrophoresis Dielectrophoresis DNA electrophoresis Electro blotting Electro focusing Gel electrophoresis Immunoelectrophoresis Isotachophoresis Pulsed field gel electrophoresis

8. Function of buffer 1. carries the applied current 2. established the pH 3. determine the electric charge on the solute High ionic strength of buffer produce sharper band produce more heat Commonly used buffer Barbital buffer & Tris-EDTA for protein Tris-acetate-EDTA & Tris-borate-EDTA (50mmol/L; pH 7.5-7.8) Buffers

9. Protein Electrophoresis Sample preparation Two-dimensional electrophoresis Detection of spots Image analysis Spot excising Enzymatic digestion of proteins Mass spectrometry Bioinformatics

10. Sample Preparation Cell washing Cell disruption Protein precipitation Solubilization Protection against protease activities Removal of nucleic acids lipids salts , buffers, ionic small molecules insoluble material

11. Cell Washing To remove contaminant material. Frequent used buffer PBS (phosphate buffer saline ): sodium chloride, 145 mM (0.85%) in phosphate buffer, 150 mM, pH7.2 Tris buffer sucrose (10mM Tris, 250 mM sucrose , (pH 7,2) Enough osmoticum to avoid cell lysis

12. Cell Disruption Methods Gentle lysis method 1. Osmotic lysis (cultured cells) Suspend cells in hypo-osmotic solution. 2. Repeated freezing and thawing (bacteria) Freeze using liquid nitrogen 3. Detergent lysis (yeast and fungi) Lysis buffer (containing urea and detergent) SDS (have to be removed before IEF) 4. Enzymatic lysis (plant, bacteria, fungi) Lysomzyme (bacteria) Cellulose and pectinase (plant) Lyticase (yeast)

13. Cell disruption (continued) Key variable for successful extraction from crude material 1. The method of cell lysis 2. The control of pH 3. The control of temperature 4. Avoidance of proteolytic degradation

14. Removal of Contaminants Major type of contaminants: 1. DNA/RNA 2. Lipids 3. polysaccharides 4. Solid material 5. Salt

15. Protein Precipitation Ammonium sulfate ( salting out) TCA precipitation Acetone and/or ethanol TCA plus acetone Not efficient, de-salting neces sary Can be hard to resolubilize Leaves SDS behind, but many proteins not precipitated More effective than either alone , good for basic proteins

16. Protein Solubilization Urea (8-9.8 M) , or 7 M urea / 2 M thiourea Detergent (CHAPS,…) Reductant (DTT, 2-mercaptoethanol) Carrier ampholytes (0.8 % IPG buffer) Sonication can help Solubilization Sample can be heated only prior to addition of urea

17. Staining Methods Colloidal Coomassie stain (Gel Code Blue from Pierce) Fluorescent stain (Molecular Probes) Sypro Ruby Difference Gel Electrophoresis (DIGE) Coomassie stain Silver stain

18. 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE)

19. Protein Identification by MALDI-TOF

20. SDS-PAGE ( Sodium dodecyl sulphate polyacrylamide gel electrophoresis)

21. Procedure Protein sample is first boiled for 5 mins. in a buffer solution containing SDS and β - mercaptoethanol Protein gets denatured and opens up into rod-shaped structure. Sample buffer contains bromophenol blue which is used as a tracking dye, and sucrose or glycerol. Before the sample is loaded into the main separating gel a stacking gel is poured on top of the separating gel.

22. Procedure continued… Current is switched on. The negatively charged protein-SDS complexes now continue to move towards the anode. As they pass through the separating gel, the proteins separate, owing to the molecular sieving properties of the gel. When the dye reaches the bottom of the gel, the current is turned off. Gel is removed from between the glass plates and shaken in an appropriate stain solution. Blue colored bands are observed under UV rays.

23. Choice of Electrophoretic System

24. Choice of Electrophoretic System

25. Zonal Electrophoresis

26. Application of Electrophoresis Clinical diagnosis SDS –PAGE : used for finding molecular weight of proteins and their purification . Separation and analysis of nucleotides and nucleic acids, DNA finger printing. Hemoglobin separation Lipoprotein separation and identification Determination of molecular weight of proteins .

27. References Dunbar, B. S. (1987). Two Dimensional Electrophoresis and Immunological Techniques. New York: Plenum Press . Gersten , D. M. (1996). Gel Electrophoresis ( Essential Techniques Series). New York: Wiley. Kuhr , W. G. (1990). Capillary Electrophoresis. Anal Chem 62 :403R–414R. Laemmli , U. K. (1970). Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 . Nature 227 :680. Sambrook , J., Fritsch, E. F., and Maniatis , T. (1989 ). Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Tal, M., Silberstein, A., and Nusser , E. (1980). Why Does Coomassie Brilliant Blue Interact Differently with Different Proteins? J Biol Chem 260 :9976–9980.Press .

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