Protein Protocols Protein Protocols

CAT Gel Electrophoresis 91
3. CAT separating gel buffer: Prepare a 1.5 M Tricine-NaOH solution by dissolving 134.4 g
of Tricine in 400 mL of distilled water. Add NaOH until the pH of the solution reaches
8.0. Bring the solution to a total volume of 500 mL using distilled water. As with all
Tricine solutions, the pH of CAT separating gel buffer should be determined at the
expected temperature of use. The CAT separating gel buffer should be stored at room
temperature to avoid any precipitation that may occur during long-term cold storage.
4. CAT sample buffer: Dilute 0.67 mL of CAT separating gel buffer to approx 80 mL with
distilled water; to this add 10 mL of glycerol and 1 g of CTAB. Mix the solution until all
the components are dissolved, and adjust the pH to 8.8 using NaOH. Bring the solution to
a final volume of 100 mL using distilled water. In some cases, it may be helpful to add a
low-mol-wt cationic dye that will be visible during electrophoresis: 10 µL of a saturated
aqueous solution of crystal violet may be added/mL of sample buffer. Note that CTAB is
corrosive, and care should be taken when handling CTAB powder or CTAB solutions:
avoid inhalation or skin contact as advised by the supplier. Store CAT sample buffer at
room temperature to avoid precipitation of the components.
5. Acrylamide stock solution: A 40% acrylamide stock solution may be prepared by combining
38.93 g of ultrapure acrylamide with 1.07 g of bis-acrylamide in a total of 100 mL of
distilled water. The final solution is 40%T (w/v) and 2.67%C (w/w). The “%T” and “%C”
values indicate that the total amount of acrylamide in solution is 40 g/100 mL and that
the amount of bis-acrylamide included is 2.67% of the total acrylamide by weight. The
acrylamide stock solution should be stored in the refrigerator. Unpolymerized acrylamide
is very toxic, and great care should be taken when handling acrylamide powders and solutions:
Follow all precautions indicated by the supplier, including the wearing of gloves
and a particle mask during preparation of acrylamide solutions.
6. Agarose stock solution: A ready-to-use agarose stacking gel solution may be prepared by
combining 25 mL of CAT stacking gel buffer, 0.1 g CTAB, and 0.7 g of electrophoresisgrade
agarose distilled to a final volume of 100 mL. Mix the components well, and, if
necessary, adjust the pH to 10.0. Heat the solution in a microwave oven to melt the agarose,
and swirl the solution to mix thoroughly. Divide the agarose stock solution into 10
aliquots, and store at 4°C until ready to use.
7. 10% Ammonium persulfate (AP): Dissolve 0.1 g of ammonium persulfate in 1 mL of
distilled water. Make just prior to use.
8. Water saturated isobutanol: Combine equal volumes of isobutanol and distilled water.
Mix well, and allow the two phases to separate: the water-saturated isobutanol will be the
upper layer. Store at room temperature in a clear container so that the interface is visible.
9. CAT gel fixative: Combine 40 mL of distilled water, 10 mL of acetic acid, and 50 mL of
methanol; mix well. Store CAT gel fixative in a tightly sealed container at room temperature.
10. Coomassie brilliant blue stain (CBB): Combine 40 mL of distilled water with 10 mL of
acetic acid and 50 mL of methanol. Add 0.25 g of CBB R-250, and dissolve with stirring
(usually overnight). Filter the solution through #1 Whatman paper to remove any particulate
material. Store at room temperature in a tightly sealed container.
11. CBB Destain: Combine 437.5 mL of distilled water, 37.5 mL of acetic acid, and 25 mL of
methanol. Mix well, and stored in a closed container at room temperature.
12. Electrophoresis apparatus: A suitable electrophoresis apparatus and power supply are
required to run CAT gels. It is desirable to set aside combs, spacers, gel plates, and buffer
tanks to use specifically with CAT gels; however, if the same apparatus is to be used
alternately for CAT gels and SDS gels, it is necessary to clean it thoroughly between each
use. Often, the first CAT gel run in an apparatus dedicated to SDS gels will have a smeared
appearance with indistinct bands. This smearing is the result of residual SDS, and subse-