Protein Protocols Protein Protocols

234 Springer
Table 1
Recipe for Various Percentages of SDS-PAGE Gels
3. At this point, the gel can be rinsed in glass-distilled water for 2 h to overnight (see Note 2).
4. After the water rinse, add fresh water and enough crystalline dithiotreitol (DTT) to make
the solution 5 µg/mL.
5. After 15 min, remove the DTT, and add 0.1% silver nitrate made fresh from the 1% stock
solution. Shake for 15 min.
6. Rapidly rinse the gel with a small amount of water followed by two 5–10 mL rinses with
developer followed by the remainder of the developer.
7. Watch the gel carefully, and add stop bath to the gel and developer when the desired
darkness of the bands is reached.
8. Store the stopped gel in water until the next step.
3.3. Slicing and Counting of Gel Slices
Percentage of acrylamide
7.5 10 12.5
Stock solution
Acrylamide-DATD, mL 0.96 1.33 1.66
Tris II, mL 1.50 1.50 1.50
Water, mL 3.50 3.13 2.80
APS, mL 0.03 0.03 0.03
TEMED, mL 0.003 0.003 0.003
1. Remove individual lanes from the gel for slicing by cutting with a knife or spatula.
2. Cut each lane into uniform slices, or cut identified bands in the gel (see Note 3).
3. Place each slice into a glass scintillation vial, and add 0.5 mL of 2% sodium metaperiodate
solution.
4. Shake the vials for 30 min to dissolve the gel.
5. Add a 10-mL aliquot of scintillation fluid to the vial, cool the vial, and count in a refrigerated
scintillation counter (see Notes 4–6).
4. Notes
4.1. SDS-Polyacrylamide Gels
1. Acrylamide-DATD gels behave quite similarly to acrylamide-bis gels, except for the fact
that for a given percentage of acrylamide, the relative mobility of all proteins are reduced
in the DATD gel. In other words, a DATD gel runs like a higher percentage acrylamidebis
gel.
4.2. Silver Staining
2. The water rinse can be reduced to 1 h, if the water is changed at 10–15 min intervals. The
author found in practice that the amount of DTT was not particularly critical, and routinely
added the tip of a microspatula of crystals to approx 30 mL of water.
4.3. Slicing and Counting of Gel Slices
3. The method seems relatively insensitive to gel volume, as measured by changes in efficiency
(3) over the range of 5–100 mm3 for tritium and an even larger range for 14C or 35S.