2.1. Cell Culture and Detergent Extraction Buffers
1. Dulbecco's Modified Eagle's Medium (DMEM; Invitro-gen/Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, HyClone, Logan, UT) and 1/200 dilution (v/v) of penicillin/streptomycin stock solution (10,000 units/mL;10,000 mg/ml) (Invitrogen/Gibco, Grand Island, NY).
2. Solution of trypsin (0.05%) and ethylenediamine tetraacetic acid (EDTA; 1 mM) from Invitrogen/Gibco.
3. Opti-MEM serum free transfection media and Lipofectamine 2000 reagent (Invitrogen/Gibco).
4. Piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) 4X stock buffer: 10 mM PIPES, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl2 (see Note 1).
5. 100 mM EDTA stock solution: Dissolve 3.6 g of EDTA in 100 mL of water (final volume). Store at room temperature.
6. 100 mM phenylmethylsulfonyl fluoride (PMSF) stock solution: Dissolve 174 mg of PMSF in 10 mL of isopropanal and vigorously vortex. Store at room temperature in the dark.
7. Modified digitonin extraction buffer for cytosolic fractionation (11). 10 mM PIPES, pH 6.8; 0.015% (w/v) digitonin; 300 mM sucrose; 100 mM NaCl; 3 mM MgCl2 ; 5 mM EDTA; 1 mM PMSF; and 40 mM iodoacetamide (IA). Add 25 mL of 4X PIPES stock buffer and 60 mL of water in a small flask with a stir bar and dissolve 18.75 digitonin powder by heating. Once dissolved, add 5 mL of EDTA stock buffer and 1 mL of PMSF stock buffer. Cool the solution to 4°C, adjust pH to 6.8, and bring final volume up to 100 mL with water. The extraction buffer should be aliquoted, fresh-frozen, and stored at -80°C until use. (see Note 2).
8. Modified Triton X-100 extraction buffer for membrane/ organelle fractionation (11). 10 mM PIPES, pH 7.4; 0.5% (v/v) Triton X-100; 300 mM sucrose; 100 mM NaCl; 3 mM MgCl2; 5 mM EDTA, 1 mM PMSF; and 40 mM IA. Add 25 mL of 4X PIPES stock buffer, 1 mL of PMSF stock buffer,
2.2. Redox Two-Dimensional Polyacrylamide Gel Electrophoresis (Redox 2D-PAGE)
3 mL of EDTA stock buffer, and 5 mL of freshly prepared 10% (v/v) Triton X-100. Cool to 4°C, adjust pH to 7.4 and add ultrapure water to a final volume of 100 mL. Aliquot and store at -80°C. (see Note 2).
9. SDS nuclear extraction buffer: 50 mM Tris-HCl, pH 7.4; 2%% (w/v); 1 mM PMSF; 40 mM IA. Solution should be made fresh for each experiment and kept at room temperature until use to prevent precipitation of SDS.
10. Trichloroacetic acid (Sigma-Aldrich, St. Louis, MO) 100% (w/v) for protein precipitation (avoid contact with skin). Dissolve in ultrapure water and store at 4°C.
11. Protein resolubilization buffer: 100 mM Tris-HCl, pH 8.0; 2%% SDS; 1 mM PMSF; and 40 mM IA. Make fresh as required.
12. Lowry detergent compatible protein assay (BioRad, Hercules, CA).
1. Solutions for Tris/glycine SDS PAGE: 30% acrylamide/bis solution (37.5:1 with 2.6% C) (avoid skin contact by wearing gloves when handling) and N,N,N,N'-tetramethyl-ethylen-ediamine (TEMED, BioRad). 1 M Tris-HCl (pH 6.8), 1.5 M Tris-HCl (pH 8.8), and 10%% SDS (w/v); store at room temperature. Ammonium persulfate (APS, BioRad): prepare 10% (w/v) fresh in ultrapure water as required.
2. Running buffer (10X): 240 mM Tris-Base, 192 mM glycine, and 1% (w/v) SDS. Dissolve 29 g of Tris-base, 144 g of glycine, and 10 g of SDS in 800 mL of ultrapure water then adjust final volume to 1,000 mL. 1X running buffer should be approx pH 8.3. Store at room temperature.
3. 2X SDS sample buffer: 80 mM Tris-HCl, pH 6.8; 4% SDS; 20% glycerol; and 0.01% (w/v) bromophenol blue. Make a 200-mL volume, filter sterilize, and store at room temperature.
4. Agarose overlay solution. Make 10 mL of 2% (w/v) low-melt agarose (BioRad) in 1X running buffer fresh by microwav-ing for 30 s and let cool for several minutes before using.
5. Prestained molecular weight markers. Precision Plus Protein All Blue Standards (BioRad).
6. 1 MDL-dithiothreitol (DTT) stock solution. Dissolve 1.54 g of DTT in 10 mL of water, aliquot in 1-mL volumes, and store at -20°C.
7. No. 9 single edged industrial razor blades (Fisher).
1. Mass spectrometry (MS)-compatible silver stain: (a) Fixer: 50% methanol, 5% glacial acetic acid; (b) Sensitizer: 0.02% (w/v) sodium thiosulfate (Na2 S2O3); (c) Silver Stain: 0.1% (w/v) silver nitrate (AgNO3 ); (d) Developer: 0.04% (v/v)
2.4. Mammalian Expression Vectors and Immunoprecipita-tion Buffer
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