Electrophoresis Theory

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Electrophoresis Theory
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Gel Electrophoresis

• friction is ease at which mole-cule passes
  through pores
• size is the major determinant

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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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)
mobility ? (voltage) (mass)
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Size Standards

• 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

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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
• ? 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 (EIR)

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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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