Protein Protocols Protein Protocols

CAT Gel Electrophoresis 93
Table 1
Preparation of Acrylamide Solutions for CAT Gels
Regent 4%T, mL 6%T, mL 8%T, mL 10%T, mL
40%T Acrylamide 1.00 1.50 2.00 2.50
Tricine buffer 2.50 2.50 2.50 2.50
Distilled water
Degas solution
6.39 5.89 5.39 4.89
10% AP 0.10 0.10 0.10 0.10
TEMED 0.01 0.01 0.01 0.01
Volumes indicated are in milliliters required to prepare 10 mL of the desired
solution. Solutions should be degassed prior to the addition of the crosslinking
agents, AP and TEMED.
5. Carefully pour the gel mixture into the gel plates to the desired volume; remember to
leave room for the stacking gel and comb.
6. Finally, layer a small amount of water-saturated isobutanol onto the top of the gel. The
isobutanol layer reduces the penetration of atmospheric oxygen into the surface of the gel
and causes the formation of an even gel surface. Allow polymerization of the separating
gel to proceed for at least 60 min to assure complete crosslinking; then pour off the
isobutanol, and rinse the surface of the separating gel with distilled water.
3.3. Casting CAT Stacking Gels
Two different types of gel stackers are routinely used with CAT gels. For gel histochemical
analyses, or where subsequent protein activity assays will be performed,
stacking gels made from agarose have provided the best results.
1. Slowly melt a tube of agarose stock solution in a microwave oven; avoid vigorous heating
of the solution, since boiling will cause foaming to occur and may result in air pockets in
the finished gel.
2. Insert the gel comb into the apparatus, and cast the stacking gel directly onto the surface of
the acrylamide separating gel. Allow the agarose to cool thoroughly before removing the
comb (see Note 8).
3. As an alternative to agarose stacking gels, low%T acrylamide stackers may also be used.
To prepare an acrylamide stacking gel, combine the 40%T acrylamide stock, CAT
separating gel buffer (0.5 M Tricine-NaOH, pH 10.0), and distilled water in the ratios
indicated in Table 1. Typically, a 4%T stacking gel is used. Degas the solution by applying
a moderate vacuum for 5–10 min. Next, add 10% AP and TEMED to the solution as
indicated in Table 1, and swirl the solution gently to mix. Insert the gel comb and cast the
stacking gel directly onto the surface of the acrylamide separating gel. Do not use watersaturated
isobutanol with stacking gels! It will accumulate between the comb and the gel,
and cause poorly defined wells to form. Allow the stacking gel to polymerize completely
before removing the comb.
3.4. Loading and Running CAT Gels
1. After the stacking and separating gels are completely polymerized, add 1X CAT tank
buffer to the gel apparatus so that the gel wells are filled with buffer prior to adding the
samples.