Electrophoresis, protein separation, Bradford assay

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Life Science
LIFE 212
Farida Safadi- Chamberlain

26 September Part 1: Electrophoresis Greek – phoresis: transmission Gel electrophoresis: A technique of molecular separation The movement of charged molecules in an electric field Molecules separate in a porous matrix (gel) according to their charge, size and shape Proteins are charged molecules Proteins are chains of amino acids Types of protein separation 1. Native gel electrophoresis: proteins separate by size, charge, and conformation Uses: isozymes, enzyme activity 2. Isoelectric focusing: proteins separate by their isoelectric point (pI) in a pH gradient pI: the pH at which protein net charge is zero 3. SDS – Denatured Gels: separate proteins according to size (MW) Boiling: breaks H-bonds SDS: breaks noncovalent bonds, coats with –ve charge β-Mercatoethanol: S-S → SH Migration of denature proteins: inversely proportional to their MW 4. 2D gels: 2 & 3 High resolution separation Used in proteomics analyses Types of gels Tube gels Slab gels Continuous gels Discontinuous gels Discontinuous gel electrophoresis system: Components Resolving gel (12% polyacrylamide) Stacking get (4% polyacrylamide) Lower electrode buffer (lower chamber) Upper electrode buffer (upper chamber) Protein sample Anode & cathode Part 1: Gel Electrophoresis Separation of partially purified potato tyrosinase on SDS denaturing gel electrophoresis 2X Sample Buffer Buffer: Tris-HCl, pH 6.8 Glycerol 10% SDS: detergent Bromophenol blue β-Mercaptoethanol Why Glycerol? Keeps it in wells Makes it heavy Why SDS? Opens tertiary structure of proteins Four factors are responsible for tertiary structure Disulfide linkages Hydrogen bonding Electrostatic interactions Hydrophobic interactions Breaks hydrophobic interactions, adds –ve charge Why β-Mercaptoethanol: S-S → SH Why bromophenol blue? Can see that it’s loaded in wells Gel Electrophoresis: Determining Protein Molecular
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