Protein Protocols Protein Protocols

1102 Johansen and Svensson
Table 4
Polyacrylamide Gel Mixtures
Separation gela 6% 7.5% 10% 11% 15%
40% Acrylamide 6 mL 7.42 mL 10 mL 11 mL 15 mL
2% bis 3.24 mL 4 mL 5.3 mL 5.88 mL 10 mL
8X 1 M Tris (pH 8.8) 4.9 mL 4.9 mL 4.9 mL 4.9 mL 4.9 mL
dH2O 25.22 mL 22.77 mL 19 mL 17.5 mL 9.48 mL
10% Amps 230 µL 230 µL 138 µL 138 µL 138 µL
10% SDS 0.4 mL 0.4 mL 0.4 mL 0.4 mL 0.4 mL
TEMED 33 µL 33 µL 14 µL 14 µL 14 µL
Stacking gel b 3.5% 4.5%
40% Acrylamide 1.3 mL 1.68 mL
2% bis 1 mL 0.9 mL
8X 1 M Tris (pH 6.8) 1.9 mL 1.9 mL
dH20 11 mL 10.5 mL
10% Amps 110 µL 100 µL
10% SDS 150 µL 150 µL
TEMED 20 µL 20 µL
a Total volume 40 mL.
b Total volume 15 mL.
detection level 10-fold. Several commercial preparations are available but in most
cases 1 M sodium salicylate (pH 6.0) treatment for 30 min will serve the purpose.
2. Materials
2.1. Metabolic Labeling of Proteins
1. Monolayer cultures approximately subconfluent to confluent or suspension cultures.
2. Methionine-free and/or cysteine-free medium.
3. 35S methionine and/or 35S cysteine. The rate of synthesis and the half-life of the protein of
interest as well as the number of cells to be labeled affect the time required as well as the
intensity of labeling. Labeling for 2–4 h with 100–400 µCi of 35S-labeled amino acids
is commonly used, preferably at the time of maximum protein synthesis in the cells
(see Note 1).
2.2. Lysis of Cells
Choose one of the following lysis buffers (1–6) (see Note 3):
1. Triple-detergent lysis buffer: 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.02% sodium
azide, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate.
2. Single-detergent lysis buffer: 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.02% sodium
azide, 1% Triton X-100 or NP-40.
3. High salt lysis buffer I: 50 mM HEPES (pH 7.0), 500 mM NaCl, 1% NP-40.
4. High salt lysis buffer II: 10 mM Tris-HCl (pH 7.8), 150 mM NaCl, 600 mM KCl, 5 mM
EDTA, 2% Triton X-100.
5. No salt lysis buffer: 50 mM HEPES (pH 7.0), 1% NP-40.