Exercise 8 - EXERCISE 8/CHAPTER 8 PROTEIN ELECTROPHORESIS...

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EXERCISE 8/CHAPTER 8 PROTEIN ELECTROPHORESIS AND ELECTROBLOTTING Electrophoresis separates molecules into groups based on size or net charge; gives information about the native state of the molecule and partially purify that molecule The gel acts as a sieve through which the molecules and ions pass, hence the pore size in the gel determines how far molecules can move into the gel Electrophoresis Separates Molecules By Size and Charge Gel Electrophoresis: ions and molecules dissolved in a buffer solution move through a semi-solid gel in response to an electrical field Can also be performed using paper or silica-based thin layer chromatography plates as medium Has an anode (+ end that attracts anion) and a cathode (- end that attracts cations) Electrical field is created, usually through a buffer solution that permeates the gel Preparation of Gels for Electrophoretic Separation of Proteins Polyacrylamide Gel Electrophoresis (PAGE) : polyacrylamide polymerizes into a mesh-like matrix, the pore size can be controlled during formation of gel Acrylamide : small, water-soluble organic molecule that combines end-to-end chemically with another identical acrylamide molecule (making a dimer); these combine to eventually form polyacrylamide BIS: acrylamide-like molecule that combines with 2 polyacrylamide molecules, linking them together Acrylamide and BIS only readily form polymers when a few free radicals (unpaired e-) are present in the solution o free radicals are produced by specific reaction involving ammonium persulfate (APS), which dissociate into sulfate free radicals dissolve in water in the presence of TEMED Sulfate and TEMED free radicals react with and activate an acrylamide or BIS molecule, enlarging the molecule until a large polymer is formed Solutions = 3% - 20% acrylamide; ratio of 40 acrylamide to 1 BS o Concentration of BS affects pore size of gel Principles of Gel Electrophoresis: 2 opposite ends come into direct contact with separate reservoirs of liquid (buffer) Molecules move through gel depending on size, shape, and net charge Each discrete band may be composed of many different types of proteins, all of which migrated to the same location in the gel due to the particular combination of characteristics that each has PAGE is an empirical technique; it does not predict accurately the relative rates of migration through a gel except by observation Net negative charge of protein increases as pH of its surrounding solution increases and vice versa
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Role of Charge Carriers in Electrophoresis: o Current is carried through gel in form of electrical charge carriers, which are primarily the smallest charged species that are available. (ex: Cl-) o Order of materials in gel after being electrophorized: Glycine (uncharged): small amino acid in the buffer solution; does not migrate Proteins (-): sandwiched between bromophenol blue and glycine band Bromophenol blue (-):
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This note was uploaded on 03/20/2008 for the course BIO 205L taught by Professor Hanson during the Fall '07 term at University of Texas.

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Exercise 8 - EXERCISE 8/CHAPTER 8 PROTEIN ELECTROPHORESIS...

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