Materials

All solutions should be made of Analar-quality reagents and distilled water. Stock solutions of 20% (w/v) SDS, phosphate-buffered saline (PBS), 5 M NaCl, 0.5 M EDTA pH 8.0, 1 M dithithrietol, 1 M Tris HCl at pH 8.8, 8, 7.5, and 6.8 are required.

1. Lysis buffers:

a. RIPA lysis buffer: 150 mM NaCl, 1% (v/v) NP-40, 0.5% (w/v) deoxycho-late, 0.1% (w/v) SDS, 50 mM Tris-HCl, pH 8.0, and protease inhibitors. NP-40 lysis buffer: 150 mM NaCl, 1% (v/v) NP-40, 50 mM Tris, pH 7.5, and protease inhibitors.

b. Protease inhibitors: A mixture of 1 mM phenylmethylsulfonyl fluoride (PMSF), (the 1 M concentrated stock dissolved in acetone), and 1 mg/mL each of antipapain, chymostatin, leupeptin, and pepstatin (see Note 1).

c. 2X dissociation buffer: 4% (w/v) SDS, 120 mM Tris-HCl, pH 6.8, 0.002% (w/v).

d. Bromophenol blue, 20% (v/v) glycerol 0.1 M dithiothreitol (see Note 2).

2. Antibody coupling solutions: 0.1 M borate buffer pH 8.2, 20 mM ethanolamine pH 8.2, 0.2 M triethylamine, pH 8.2, dimethyl pimelimidate, and protein A Sepharose beads (Amersham Pharmacia Biotech, Little Chalfont, UK).

3. Kinase buffer: 100 mM NaCl, 25 mM HEPES pH 7.5, 5 mM MnCl2, 10 mM MgCl2, 1 mM Vanadate, 10 |M Mg-ATP (Sigma), y32P-ATP (3000 Ci/mmol).

4. SDS polyacrylamide gel electrophoresis (PAGE) solutions.

a. Resolving gel: 12.5 mL 30% (29:1) acylamide/fci's-acrylamide mix, 7.5 mL 1.5 M Tris-HCl, pH 8.8, 0.3 mL 10% (w/v) SDS, 9.9 mL H2O, 0.3 mL 10% (w/v), ammonium persulfate (made fresh), 12 |L TEMED.

b. Stacking gel: 0.83 mL of 30% (29:1) acylamide/fci's-acrylamide mix, 0.63 mL 1M Tris-HCl, pH 6.8, 50 |L 10% (w/v) SDS, 3.4 mL H2O, 50 |L 10% (w/v) ammonium persulfate, 5 |L TEMED.

Running buffer: 25 mM Tris base, 192 mM glycine, 0.1% (w/v) SDS.

5. Blotting solutions:

a. Semidry blotting buffer: 48 mM Tris base, 39 mM glycine, 0.037% (w/v) SDS, 20% (v/v) methanol.

b. Western blocking buffer: PBS 5%(w/v), BSA 0.05% (v/v), Tween-20, or PBS, 5% (w/v) nonfat milk powder.

c. Western washing buffer: PBS 0.05% Tween-20.

d. ECL reagent commercially available from Amersham Pharmacia Biotech.

6. Antibody-labeling solutions:

25 mM KI, Iodogen (Pierce, Warriner, UK), CHCl3, Sephadex G-25

(Amersham Pharmacia Biotech), and 125I (100 mCi/mL).

7. Gel-staining solutions:

a. Gel-fixing solution: 45% (v/v) methanol, 9% (v/v) acetic acid.

b. Coomassie stain: 45% (v/v) methanol, 9%(v/v) acetic acid containing 0.25% (w/v) Coomassie blue R250.

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