230-7-ProtElec - Bio 230 Electrophoresis of proteins...

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Bio 230 Electrophoresis of proteins INTRODUCTION In lecture you were introduced to some characteristics and functions of proteins. In previous labs you have investigated enzyme activity, performed a crude protein extraction, and measured protein concentration. In this lab you will be introduced to electrophoresis, a widely used technique for analyzing proteins and nucleic acids. You will use acrylamide gels to separate proteins by mass (molecular weight) now, and later in the semester you will use agarose gels to separate DNAs by length (approximate numbers of nucleotides). Electrophoresis separates charged molecules and requires a power supply to provide negatively charged electrons, a plastic tank, and a solution of ions. Electrons enter the tank at the negatively charged cathode (always black). Electrons leave the tank at the positively charged anode (always red). The flow of electrons sets up a potential, or energy difference between the electrodes, measured in volts. The flow of electrons (current) is measured in milliamperes. Ions in the tank will flow in one direction or the other, according to their charge. DNA and RNA are uniformly negatively charged (due to their phosphate groups). Proteins are more complicated and may have a net positive, negative or zero charge. (You should be able to explain why.) Electrophoresis is performed through a gel that acts as a molecular sieve hindering the flow of molecules in the electric field. Shorter or more compact molecules travel more rapidly through the mesh than longer or more floppy molecules. If similar sized molecules have different charges they will also travel at different rates. Gels must be run long enough that molecules are resolved into separate bands different distances from the origin, but not so long that everything runs off the end of the gel. Two common gel materials used in cell biology are agarose and polyacrylamide. Agarose is a derivative of agar, the extract from seaweed used for growing bacteria in petri dishes. Agarose forms a gel after heating and cooling (just as gelatin does). Polyacrylamide is formed by chemical cross-linking of acrylamide and bisacrylamide. Acrylamide monomer is a neurotoxin, but in the polymerized form it is not. To separate negatively charged molecules you load them at the cathode, and they are driven from the cathode toward the anode. Positively charged molecules move in the opposite direction, so to separate them you must load at the anode. If you have an agarose gel with loading wells in the middle you can run a mixture of positive and negative molecules and they will go in opposite directions! Uncharged molecules do not move at all. The speed at which a molecule travels between electrodes depends on the type and concentration of gel material, the molecule’s charge, shape, and size. Higher percentages of agarose or acrylamide separate smaller molecules; lower percentages separate larger molecules. Molecules with the same mass may be separated by differences in charge. Molecules
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230-7-ProtElec - Bio 230 Electrophoresis of proteins...

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