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Electrophoresis Theory. v = (E/d)(q)/(6 r ) mobility electric field strength net charge shape viscosity size mobility = (applied voltage)(net charge)

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Presentation on theme: "Electrophoresis Theory. v = (E/d)(q)/(6 r ) mobility electric field strength net charge shape viscosity size mobility = (applied voltage)(net charge)"— Presentation transcript:

1 Electrophoresis Theory

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

3 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)

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

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

6 Slab Gels

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

8 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)

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

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

11 Practical Considerations prepare gels choose % acrylamide prepare samples stacking gel buffer + 2% SDS -mercaptoethanol heating (37 o 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)

12 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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