electrophoresis theory. v = (e/d)(q)/(6 r ) mobility electric field strength net charge shape...
TRANSCRIPT
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Electrophoresis Theory
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v = (E/d)(q)/(6r)mobility
electric field strength
netcharge
shape viscosity
size
mobility = (applied voltage)(net charge)
(frictional coefficient)
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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)
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Polyacrylamide Gels
• common matrix for gel electrophoresis
• agarose has larger average pore size
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Basic Apparatus
•gel is placed between electrodes
•buffers complete the circuit
•proteins loaded onto top of gel
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Slab Gels
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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
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• 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)
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•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
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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
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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)
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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
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