Download charge-to-mass ratio. The electrophoretic mobility is defined as the

Purification of Proteins
Page 3 of 3
media (similar to gel filtration). Therefore, the electrophoretic mobility of
proteins in gels will depend on both the size as well as the charge-to-mass ratio.
Therefore, to separate proteins according to size it is necessary to give them to
same charge-to-mass ratio. This can be accomplished by denaturing the protein in
SDS (sodium dodecyl sulfate, shown below).
The SDS binds to most proteins in a uniform manner giving all proteins the same
charge-to-mass ratio.
The electrophoretic mobility is defined as the distance migrated in a certain time
period. Thus the mobility is directly proportional to the velocity. The effect of the
gel matrix is to produce the following dependence of the molecular weight on the
mobility.
log(M ) = c*Mobility + d
r
d is the distance between the bottom of the well in which the protein is applied, and the top of the resolving gel
Where "c" is a proportionality constant that depends on the gel properties. Each
gel has a region where the above equation holds and a plot of log(MW) vs.
Mobility will be linear. The determination of molecular weight requires the
calibration of the SDS gel using proteins of known molecular weight. Note the
similarity between the above equation and that used for gel filtration. For
example, if hemoglobin were run as a standard, it would result in a band on the
gel at a mobility corresponding to Mr = 16 kDa, i.e. its monomer molecular
weight and myoglobin (Mr = 17 kDa) would be nearby because it is a singlesubunit protein.
(See the second Example Graph for the similarity in the relative mobilities.)
Isoelectric Focusing (IEF)
In this technique a uniform solution of protein is mixed with a special polymer.
The properties of this polymer are such that they will form a pH gradient when an
electric field is applied across the solution (the various species of the polymer
migrate in solution until they reach their isoelectric point). While this pH gradent
is being formed the protein molecules also migrate until they reach their
isoelectric point. This is a convenient way to obtain the isoelectric point of a
protein as well a to purify complex mixtures of proteins.
Detection of Separated Proteins in SDS-PAGE or IEF
1. Coomassie blue (blue dye that binds to proteins)
2. Fluorescamine (fluorescent compound that reacts with proteins)
3. Autoradiography (detection of radioactivity in proteins)
4. Immunoblots (use of an antibody against the protein to localize it)
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10.3.02
http://www.bio.cmu.edu/Courses/03231/LecF02/Lec16/lec16.html
2/6/2004