Electrophoresis Theory

v = (E/d)(q)/(6r)mobility

electric field strength

netcharge

shape viscosity

size

mobility = (applied voltage)(net charge)

(frictional coefficient)

Gel Electrophoresis• 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

• 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

SDS-PAGE

mobility

(voltage)

(charge)

(mass)

•proteins of known mass used as standards to calibrate gels

•mobility on gels defined as Rf

•Rf = distance protein migrated length of gel •or BB dye front

x

y

Rf = x/y

Size Standards

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 -mercaptoethanol• heating (37o boil)

• electrophoresis• amount of sample• voltage• tracking dye (BB)

• 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