Protein Protocols Protein Protocols

Radiolabeled Proteins 233
9. Sample buffer: The following are mixed together and stored at 4°C: 1.5 mL 20% (w/v)
SDS, 1.5 mL glycerol, 0.75 mL 2-mercaptoethanol, 0.15 mL 0.2% (w/v) Bromophenol
blue, and 1.1 mL Tris I.
2.2. Silver Staining of Gels
All solutions must be made with good-quality water.
1. Solution A: methanol:acetic acid:water (50:10:40).
2. Solution B: methanol:acetic acid:water (5:7:88).
3. Solution C: 10% (v/v) glutaraldehyde.
4. 10X Silver nitrate: 1% (w/v) silver nitrate stored in brown glass bottle.
5. Developer: Just before use, 25 µL of 37% formaldehyde are added to freshly made 3% (w/v)
sodium carbonate.
6. Stop bath: 2.3 M Citric acid (48.3 g/100 mL).
2.3. Solubilization and Counting of Gel Slices
1. Solubilizer: 2% sodium metaperiodate.
2. Scintillation fluid: Ecolume (+) (ICN Biomedical, Inc., Irvine, CA) was used to develop
the method. See Subheading 4. Notes. for other scintillants.
3. Methods
The general requirements to construct, prepare, and run SDS-PAGE gels as described
by Laemmli (4) are given in Chapter 11. Presented here are the recipes and solutions
developed in the author’s laboratory for this particular technique. For most of the methods
described a preferred preparation of a standard gel can be substituted, except for the
requirement of the replacement of DATD for bis at a ratio of 1:10 DATD: acrylamide
in the original formulation.
3.1. SDS-Polyacrylamide Gels
Sufficient medium for one 8 × 10 × 0.75 to 1.0-cm gel can be made by combining
stock solutions and various amounts of acrylamide-DATD and water to achieve the
required percentage of acrylamide (see Note 1) by using the quantities listed in Table 1.
The stacking gel is that described by Laemmli (4) and is formed by combining 0.55 mL
of acrylamide-bis with 1.25 mL of Tris III, 3.2 mL of water, 0.015 mL of APS, and
0.005 mL of TEMED.
1. For both the running and the stacking gel, degas the solutions by applying a vacuum for
approx 30 s before adding the TEMED.
2. After the addition of the TEMED, pour the gels, insert the comb in the case of the stacking
gel, and overlay quickly with 0.1% SDS to provide good polymerization.
3. Prepare protein samples by dissolving two parts of the protein sample in one part of sample
buffer and heating to 100°C for 2 min.
4. Run gels at 200 V constant voltage for 45 min to 1 h or until the dye front reaches the
bottom of the plate.
3.2. Silver Staining
The method used is that of Morrissey (1).
1. Remove gels from the plates, and immerse in solution A for 15 min.
2. Transfer to solution B for 15 min and then solution C for 15 min, all while gently shaking.