Protein Protocols Protein Protocols

CAT Gel Electrophoresis 89
Fig. 1. Diagram of a CAT gel. CAT gels begin at the top with the anode immersed in tank
buffer and end at the bottom with the cathode immersed in additional tank buffer. The tank
buffer solution contains CTAB, Arginine, and Tricine. Between the tank buffers are the stacking
gel and the separating gel. The gels are made up of acrylamide polymers in a Tricine-
NaOH-buffered solution. Prior to electrophoresis, protein samples are solubilized in a sample
buffer that contains CTAB, to solubilize the protein sample, Tricine-NaOH, to maintain pH,
and carry current and glycerol, to increase specific gravity. Proteins solubilized in sample buffer
are typically layered under the upper tank buffer and directly onto the stacking gel. See Note 3
for a listing of some physical characteristics of the CAT gel components.
SDS gels. As shown in Fig. 2, a plot of relative migration distance, as a function of
known log M r of standard proteins, results in a straight line. Because of the consistent
relationship between M r and distance migrated, the relative molecular weights of
unknown proteins can be determined. CAT gels may be especially useful for the
assignment of M r to small proteins or for the comparison of proteins with very different
molecular weights. Second, the retention of significant levels of native activity in CAT
gels allows electrophoretic profiles to be assessed in situ for native activities without
additional steps to ensure protein renaturation (see Note 2). Taken together, these two
characteristics of CAT gels make them an attractive alternative to standard electrophoretic
systems.