Electrophoresis Theory

v = (E/d)(q)/(6r) net charge electric field strength size mobility shape viscosity size mobility = (applied voltage)(net charge) (frictional coefficient)

Gel Electrophoresis mobility  (voltage)(charge/mass) friction is ease at which mole-cule passes through pores size is the major determinant mobility = (voltage)(charge) (frictional coefficient) mobility  (voltage)(charge/mass)

Polyacrylamide Gels common matrix for gel electrophoresis agarose has larger average pore size

Basic Apparatus gel is placed between electrodes buffers complete the circuit proteins loaded onto top of gel

Slab Gels

Sodium Dodecyl Sulfate (SDS) strongly denaturing detergent disrupts 2o, 3o, and 4o structures binds and confers negative charge to protein charge is proportional to mass

SDS-PAGE proteins are unfolded (ie, random coil) ~ uniform charge/mass ratio due to SDS therefore endogenous charge and shape are not major factors mobility is inverse of mass mobility  (voltage) (charge) (mass)

Size Standards x y Rf = x/y proteins of known mass used as standards to calibrate gels mobility on gels defined as Rf Rf = distance protein migrated ¸ length of gel or BB dye front

Calculating MW plot log(mass) vs Rf of protein size standards ~ linear extrapolate unknowns relative molecular weight (Mr) some exceptions highly charged proteins some SDS-stable structures

Practical Considerations prepare gels choose % acrylamide prepare samples stacking gel buffer + 2% SDS  b-mercaptoethanol heating (37o boil) electrophoresis amount of sample voltage tracking dye (BB) detect proteins eg, Coomassie blue % acrylamide protein size range gradient gels desired resolution amount of sample Voltage  voltage =  time, but  heat resistance  during electrophoresis (E=IR)

Preparative Electrophoresis high resolution provides analytical information difficult to exploit in protein purification recovery of proteins from gels diffusion electroelution transfer to membrane immunization limited protein capacity special apparatus