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  • Net charge of protein=0,when pH = PI.
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    1. 1. BY- NEHA.S.SUTAR MSC PART -1 BIOTECNOLOGY
    2. 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. 3. METHODS FOR SEPARATING PROTEINS:  ELECTROPHORESIS. ISOELECTRIC FOCUSING.
    4. 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. 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. 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. 7.  TYPES OF ELECTROPHORESIS a. Agarose gel electrophoresis. b. Polyacrylamide Gel Electrophoresis c. SDS-PAGE. d. Isoelectric Focusing. e. Two-dimensional Electrophoresis.
    8. 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. 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. 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. 11. Figure 6-20 Apparatus for slab gel electrophoresis. Page146
    12. 12. SDS-PAGE Run
    13. 13. Figure 6-24 SDS-PAGE. Page149
    14. 14. Figure 6-25 Logarithmic relationship between the molecular mass of a protein and its relative electrophoretic mobility in SDS-PAGE. Page149
    15. 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. 16. Figure 6-26 General formula of the ampholytes used in isoelectric focusing. Page150
    17. 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. 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. 19. IEF PRINCIPLE:
    20. 20. Traditional Equipment for Isoelectric focusing (IEF): Ampholytes polyacrylamide Cathode (-) electrode solution Anode (+) electrode solution
    21. 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. 22. 2 –D ELECTROPHORESIS
    23. 23. Figure 6-27 Two-dimensional (2D) gel electrophoresis. Page150
    24. 24. Proteomics solution IEF SDS-PAGE
    25. 25. 2D Gel Analysis Software
    26. 26. Computerized imaging
    27. 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. 28. THANK YOU

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