Electrophoresis

Electrophoresis - molecules mainly according to their mass...

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Isoelectric point: pH at which a protein's net charge is zero; depends on the relative numbers of positive and negative amino acids Isoelectric focusing: gradient of pH is created in a gel, protein sample and an electric field is applied; protein(s) migrate through the gel until they reach a pH where they are uncharged; by charge repulsion from the electrodes, they are focused into a narrow band; pH where they come to rest is the pI. Electrophoresis: separates molecules according to their charge and mass ; apply a positive charge to the bottom of a gel (~ 200V) , proteins are drawn through the gel, move in “bands” at a rate that is determined by their mass and their total charge (large proteins move slower); proteins can be stained to show where they are o SDS-PAGE: the most common form of electrophoresis; high pH buffers (so that all proteins will be negative, and will migrate towards the anode at the opposite end of the gel); separates
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Unformatted text preview: molecules mainly according to their mass , because “shape” and charge differences have been negated. Sodium dodecyl sulfate (SDS): detergent; unfolds or “denatures” the protein • Denature: process in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent (e.g., alcohol or chloroform), or heat PAGE: polyacrylamide gel elecrophoresis o Two-dimensional electrophoresis: discriminates charge and mass; do isoelectric focusing in a small cylindrical gel, place that gel on top of a SDS-PAGE gel, and do electrophoresis o Dalton (Da): 1/12th the mass of a carbon 12 atom; kiloDalton (kDa) is used as the standard measure of molecular mass for proteins...
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