1.BY- NEHA.S.SUTAR MSC PART -1 BIOTECNOLOGY

2. WHY TO SEPARATE PROTEINS: TO STUDY THE STRUCTURE AND FUNCTION OF INDIVIDUAL PROTEIN. TO STUDY THE BASIC PROPERTIES THAT VARY FROM ONE PROTEIN TO OTHER. INCLUDING SIZE, CHARGE AND BINDING SITE. 3. METHODS FOR SEPARATING PROTEINS: ELECTROPHORESIS. ISOELECTRIC FOCUSING. 4. ELECTROPHORESIS Proteins are separated on the basis of their molecular mass ,when placed in an electric field.. 1st use was reported in 1937 by swedish biochemist ARNE TISELIUS. He introduced moving boundary electrophoresis - earliest analytical tecnique. Method takes place in solution.(prevent mixing of migrating proteins) Requires cumbersome apparatus with large amount of sample. Zone electrophoresis: sample is forced to move in solid support eg.filter paper,cellulose acetate or gel. Largely eleminates convective mixing of samples. Sample component migrates as discrete zones so, small amount f sample are required. PRINCIPLE: the force moving the macro molecule is the electrical potential E , the electrophoretic mobility of the molecule , (u) is the ratio of the velocity of the partical ,V to the electric potential. electrophoretic mobility is also equal to net charge of the molecule ,Z divided by the frictional coeffiecient f , thus ( u )= v/E = z/f. 5. Rate of migration depends on: o Net electrical charge of molecules. Size & shape of molecule. Electric field strength. Properties of supporting medium. Temperature of operation. 6. ELECTROPHORESIS: ADVANTAGES: PROTEINS ARE VISUALISED AS WELL AS SEPARATED. DEGREE OF PURITY OF A PARTICULAR PROTEIN, ISOELECTRIC POINT, MOL.WT, CAN BE DETERMINED. DISADVANTAGES: NOT USED TO PURIFY PROTEIN IN LARGE AMOUNT. AS IT CAN ADVERSLY AFFECT STRUCTURE AND FUNCTION OF PROTEINS. 7. TYPES OF ELECTROPHORESIS a. Agarose gel electrophoresis. b. Polyacrylamide Gel Electrophoresis c. SDS-PAGE. d. Isoelectric Focusing. e. Two-dimensional Electrophoresis. 8. Sodium dodecyl sulfate (SDS-PAGE) Native protein is unfolded by heating in the presence of -mercaptoethanol and SDS. SDS binds to the protein so that it stays in solution and denatures. Large polypeptides bind more SDS than small polypeptides, so proteins end up with negative charge in relation to their size. Thus, we can separate the proteins based on their mass. Native protein Heat + Reductant + SDS Denatured protein with bound SDS N C - - - - - - - - - - - - - - - - - 9. AFTER ELECTROPHORESIS: PROTEIN IS VISUALISED BY ADDING DYE- COOMASSIE BRILLIANT BLUE. IT BINDS AND ISOLATES PROTEIN . PROTEIN BAND FORM IN GEL IN AN DECREASING MOLECULAR WEIGHT ORDER. PRESENCE OF UNKNOWN PROTEIN CAN PROVIDE EXCELLENT MEASURE OF ITS MOL.WT. 10. Coomassie blue is triphenylmethane dye that binds noncovalently lysyl residues of proteins. its senstivity is fairly good and it is compatible with mass spectroscopy. Coomassie blue 11. Figure 6-20 Apparatus for slab gel electrophoresis. Page146 12. SDS-PAGE Run 13. Figure 6-24 SDS-PAGE. Page149 14. Figure 6-25 Logarithmic relationship between the molecular mass of a protein and its relative electrophoretic mobility in SDS-PAGE. Page149 15. Isoelectric focusing: A procedure to determine the isoelectric point (pI) of proteins. thus, a mixture of proteins can be electrophorised through a solution having a stable pH gradient in from the anode to the cathode and a each protein will migrate to the position in the pH gradient according to its isoelectric point. This is called isoelectric focusing. Ampholytes (amphoteric electrolytes)- low molecular mass (600-900D) ooligomers with aliphatic amino and carboxylic acid groups with a range of isoelectric points. Ampholytes help maintain the pH gradiennt in the presence of high voltage. Can also use gels with immobilized pH gradients - made of acrylamide derivatives that are covalently linked to ampholytes. Used with a gradient maker to ensure continuously varied mixture when the gel is made. 16. Figure 6-26 General formula of the ampholytes used in isoelectric focusing. Page150 17. METHOD: pH gradient is established in gel by addition of ampholytes which increases the pH from anode to cathode. A protein mixture is placed in a well on the gel. With an applied electric field ,proteins enter the gel migrates until each reaches its pH equivalent to its (pI). Each species of proteins is therby focussed into a narrow band about its pI. 18. sample pH 9 - pH 3 + Isoelectric focusing (1st dimension) General principle and protocol of 2-Dimension Electrophoresis MW pH gradient SDS-PAGE Ampholytes polyacrylamide 2nd dimension 19. IEF PRINCIPLE: 20. Traditional Equipment for Isoelectric focusing (IEF): Ampholytes polyacrylamide Cathode (-) electrode solution Anode (+) electrode solution 21. 2-D ELECTROPHORESIS: COMBINATION OF ISO-ELETRIC FOCUSING AND SDS-PAGE THE GEL IS THEN LAID HORIZONTALLY ON A SECOND GEL AND THE PROTEINS ARE SEPARATED BY SDS POLYACRYAMIDE GEL ELECTROPHORESIS. IN THIS TWO-DIMENSIONS GEL ELECTROPHORESIS HORIZONTAL SEPARATION REFLECTS DIFFRENCES IN pI. VERTICAL SEPARATION REFLECTS DIFFERENCES IN MOLECULAR WEIGHT..GENARATES ARRAY OF SPOTS EACH REPRESENTING A PROTEIN UPTO 5000 PROTEINS HAVE BEEN RESOLVED USING THIS TECNIQUE. THE INDIVISUAL PROTEIN SPOTS OBTAINED IN STAINED 2 D-GEL IS REMOVED WITH A SCALPEL,DE-STAINED AND PROTEIN ELUTED FROM THE GEL FRAGMENTS FOR IDENTIFICATIONS.CHARACTERISATION,BY MASS SPECTROMETRY.THE 2D ELECTROPHOTGRAM CAN BE ANALYSED BY COMPUTER AFTER THEY SCANNED & DIGITIZED. 22. 2 D ELECTROPHORESIS 23. Figure 6-27 Two-dimensional (2D) gel electrophoresis. Page150 24. Proteomics solution IEF SDS-PAGE 25. 2D Gel Analysis Software 26. Computerized imaging 27. 2D gel electrophoresis is generally used as a component of proteomics. The step used for the isolation of proteins for further characterization by mass spectroscopy. In the lab we use this technique for 2 main purposes: 1.)For the large scale identification of all proteins in a sample. 2.)Differential expression, to compare two or more samples to find differences in their protein expression. Applications 28. THANK YOU