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Electrophoresis Theory
electric field
strength
mobility
net
charge
size
v = (E/d)(q)/(6r)
shape
viscosity
(applied voltage)(net charge)
mobility =
(frictional coefficient)
Gel Electrophoresis
• friction is ease at which molecule passes through pores
• size is the major determinant
(voltage)(charge)
mobility =
(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
(charge)
mobility  (voltage)
(mass)
Size Standards
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
Rf = x/y
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