Protein Protocols Protein Protocols

92 Akins and Tuan
quent CAT electrophoretic runs will resolve protein bands distinctly. This smearing may
be somewhat avoided by soaking the gel apparatus and gel plates in CAT tank buffer prior
to a final rinse in distilled water at the final step in the cleaning process.
The selection of an electrophoresis apparatus to be used for CAT gels should be based
on a consideration of the electrical configuration of the system. Because molecular bromine
(Br 2) will form at the anode, the anode should be located away from the top of the
gel (see Note 5). In addition, it is important to realize that CAT gels are “upside-down”
relative to SDS gels: proteins migrate to opposite electrodes in the two systems. Some
electrophoresis apparatus are intentionally designed for use with SDS, and the anode (usually
the electrode with the red lead) may be fixed at the bottom of the gel, whereas the
cathode (usually the electrode with the black lead) is fixed at the top of the gel. If such an
apparatus is used, the red lead wire should be plugged into the black outlet on the power
supply, and the black lead should be plugged into the red outlet on the power supply.
Crossing the wires in this fashion ensures that the CTAB-coated proteins in the CAT
system will run into the gel and not into the tank buffer.
3. Method
The methods for the preparation and running of CAT gels are similar to other familiar
electrophoretic techniques. In this section, we will describe the basic methods for
preparing samples, casting gels, loading and running gels, visualizing protein bands,
and transferring proteins to nitrocellulose (or other) membranes. We will emphasize
the differences between CAT gels and other systems. To provide the best results, the
recommendations of the manufacturer should be followed concerning the assembly of
the apparatus and the casting of discontinuous gels.
3.1. Preparing Samples
1. Protein samples should be prepared at room temperature immediately prior to loading the
gel. Typically, tissue fragments, cells, or protein pellets are resuspended in 1.5-mL
microfuge tubes using CAT sample buffer (see Note 6). CAT sample buffer may also be
used to solubilize cultured cells or minced tissues directly. In each case, the samples should
be spun in a microfuge for 0.5 min at 16,000g to pellet any debris or insoluble material
prior to loading the gel. Good results have been obtained when the final concentration of
protein in CAT sample buffer is between 1 and 5 mg/mL; however, the preferred
concentration of protein will vary depending on the sample and the particular protein of
interest. A series of protein dilutions should be done to determine the optimal solubilization
conditions for a particular application.
3.2. Casting CAT Separating Gels
1. Assemble the gel plates and spacers in the gel casting stand as described by the manufacturer.
2. Prepare a separating gel solution by combining the 40%T acrylamide, CAT Separating gel
buffer, and distilled water in the ratios indicated in Table 1. Mix the solution by swirling
with the introduction of as little air as possible (oxygen inhibits the reactions necessary to
accomplish acrylamide polymerization, see Note 7).
3. Degas the solution by applying a moderate vacuum for 5–10 min: the vacuum generated
by an aspirator is generally sufficient.
4. Add 10% AP and TEMED to the solution as indicated in Table 1, and swirl the solution
gently to mix. Note that insufficient mixing will result in the formation of a nonhomogeneous
gel, but that vigorous mixing will introduce oxygen into the mixture.