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