Introduction:

Gel electrophoresis can be employed to separate proteins and DNA. It does so even in cases where there are no differences in charge to mass ratios! In these cases (SDS gel electrophoresis of proteins, agarose electrophoresis of DNA, and acrylamide gel electrophoresis of oligonucleotides) secondary sieving effects play a role.

The forward force:

Hydrodynamics is the study of fluid movement, in this case particles in a fluid.

It is empirical, i.e. based on observation.

In this case the force is applied via an electric field

F =(E/d)q
,p. Where:

E = voltage difference
d = distance between electrodes
q = net charge on protein = Ze


The resistive force:

The opposing force is fluid viscosity and a sieving effect that is produced by the supporting gel. It is represented by a form of Stoke's Law.

fv = (6rn)v or f = 6rn

Where:

f = frictional coefficient - related to viscosity and sieving
n = viscosity of the medium
v = velocity
r = Stoke's radius (the radius of a perfect unhydrated sphere having the same rate of movement as the unknown particle)

Steady-state conditions:

Under steady-state conditions the net force is = 0 (Otherwise the particle is accelerating!!)

(E/d)q = 6rnv ---> Solve for v:v = Eq/6rnd

Physical interpretation:

What is the physical sense of this equation?

Velocity:
Increases when the voltage or charge goes up
Decreases when the size (r) of the protein is up
Decreases when viscosity (n) increases
Decreases when voltage gradient is lowered (d) increases

Practical aspects:

Supports:

1. paper
2. starch
3. acrylamide, a synthetic compound, is polymerized in place using ammonium persulfate as the initiator and TEMED as the carrier of a free-radical polymerization.
4. agarose is a cross linked polysaccharide isolated from kelp.

Potential problems:

1. Differences in net charges between native proteins (charge to mass ratio is not constant)
2. Requirement for complete denaturation in SDS gel electrophoresis is not met
3. Requirement for constant amounts of SDS per unit protein (approx. 1 per 2 AA) or 1.4-1.5 g SDS/g protein is not met
4. No remaining disulfides (linear, unbranched polymer) = glycoproteins are also a problem.

Isoelectric focusing:

Proteins move until they are at a pH that produces an equal number of positive and negative residues on the protein. (remember the Henderson Hasselbalch equation!)

Doing isoelectric focusing crossed with SDS gel electrophoresis permits the separation of 1000 different spots, each corresponding to a different protein.

Centrifugation

Terms to review: sedimentation coefficient