Electrophoresis / SDS-PAGE

Electrophoresis The migration of proteins is affected by multiple factors involving their structural organization. Amino acids can carry either a net positive, net negative, or neutral charge depending on the combination of amino acids they contain. To make protein migration rates a function of molecular weight, it is necessary to impose a uniform shape and charge on all of the proteins in the mixture.

Why Use Polyacrylamide Gels to Separate Proteins? Smaller pore size than agarose Proteins much smaller than DNA average protein = 30-50 kD “average” DNA = >2000 kD

SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) CH2 CH3 SDS detergent negative charge added to proteins SDS

Why heat the samples? Heating the samples helps denature proteins and protein complexes, allowing the separation of individual proteins by size s-s - + SDS, heat proteins with SDS

- How does SDS-PAGE work? + Proteins (negatively charged due to SDS) move to positive electrode Proteins separate by size Smaller proteins move faster - + largest large small smallest