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Electrophoresis Theory

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Presentation on theme: "Electrophoresis Theory"— Presentation transcript:

1 Electrophoresis Theory

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

3 Gel Electrophoresis mobility  (voltage)(charge/mass)
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 2o, 3o, and 4o structures binds and confers negative charge to protein charge is proportional to mass

8 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) (mass)

9 Size Standards x y Rf = 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

10 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

11 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)

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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