2.1. Cell Culture and Cell Lysis
1. CASA medium: 6.7 g/L Yeast Nitrogen Base (Fisher) without amino acids, 20 g/L glucose (Sigma), and 1 g/L casamino acids (Fisher). Adenine, tryptophan, or uracil (5 g/L) are added as required.
2. 500 g of TCA (VWR) is dissolved in 227 mL of water to obtain a 100% solution (stored at 4°C).
4. Acid-washed glass-beads 425-600 pm in diameter (Sigma).
6. Centrifuge (Sigma 3K10).
1. Denaturing buffer: 25 mMTris-HCl, pH 8.8, for labeling by [14C]NEM and biotin-HPDP, pH 9 or 7.5, for fluorescent-dye labeling; 10 mM EDTA; 8 M urea; and 4% CHAPS. Filter (0.2 pm) and store at -20°C.
2. Iodoacetamide (IAM, Sigma), 50 mM final concentration, is added extemporaneously.
3. Dithiotreitol (DTT, Sigma), 20 mM, is added extemporaneously.
4. In the qualitative approach, N-(6-(Biotininamido)Hexyl)-3'-(2'-Pyridylthio) Propionamide (Biotin-HPDP, Pierce) is dissolved at 50 mM in dimethyl sulfoxiode (DMSO) and used at a 0.5 mM final concentration.
5. In the quantitative approach, 1 mCi (27 mM) N-[Ethyl-1-14C]-maleimide (NEM) at 37.5 mCi/mmol (Perkin Elmer) is dissolved in 2 mL of DMSO and stored at -80°C until use (2 mM final concentration).
2.3. Purification of Oxidized Protein Thiols (Biotin-HPDP-Labeled)
2.4. Bi-Dimensional Gel Electrophoresis: First Dimension
6. For fluorescent labeling, 1 mg of maleimide DYE680 or maleimide DYE780 (Dyomics) is added to 100 pL of DMSO. The stock solution is stored in the dark at -20°C.
7. AMS is 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, disodium salt.
8. Thermomixer comfort (Eppendorf).
1. Protein concentration is measured using the Micro BCA assay reagent kit (Pierce).
2. Affinity beads: Sepharose CL-4B (Sigma) and Streptavidine-Sepharose High Performance (Amersham Biosciences) are stored at 4°C. Beads are prewashed three times in water and three times in binding buffer, immediately before use.
3. Binding buffer: 25 ml Tris-HCl, pH 8.8; 50 ml NaCl; 5 mM EDTA; 4 M urea, 2% CHAPS.
4. Washing buffer no 1: 25 mM Tris-HCl, pH 8.8;; 1 M NaCl, 8 M urea; 4%% CHAPS.
5. Washing buffer no 2: 25 mM Tris-HCl, pH 8.8; 8 M urea; 4%% CHAPS.
7. Tumbling device.
1. Rehydration solution: 8 M urea, 4%% CHAPS, 20 mM DTT, 1% immobilized pH gradient buffer, bromophenol blue (0.05%% w/v).
2. pH 3-10 nonlinear Immobiline DryStrip gels (18 cm), regular strip holders (18 cm), IPGphor Isoelectrofocusing unit, and IPG buffer 3-10 nonlinear are from Amersham Biosciences.
3. RNAse A (Sigma; 10 mg/ml, i.e., 100x concentrated, stored at -20°C) is added to prevent interference with eventual residual RNAs.
2.5. Bi-Dimensional Gel Electrophoresis: Second Dimension
1. Equilibration buffer: 2% SDS, 6 M urea; 50 mM Tris-HCl, pH 8.8; 30% glycerol, bromophenol blue. This buffer is either supplemented with 50 mM DTT or 100 mM IAM, as indicated in the Methods section.
2. Electrophoresis buffer (10X): 250 mM Tris, 1.92 Mglycine, 1% SDS.
3. Stabilizing strip buffer: 0.5% (w/v) agarose in electrophoresis buffer.
4. Thirty percent acrylamide/0.4% bis solution (Interchim) (stock solution, stored at 4°C), 1.5 M Tris-HCl, pH 8.8; 10% SDS (0.45-pm filtered).
5. Ammonium persulfate (APS; Sigma), 10% solution in water stored at -20°C and N,N,N,N-tetramethyl-ethylenediamine (TEMED, Sigma).
6. 2-Isopropanol (Fluka) stored at room temperature.
7. Prestained molecular weight marker (Benchmark, Invitrogen).
8. Ettan DALTsix 1 mm Gelcaster, electrophoresis unit and electrophoresis power supply (EPS 601) are from Amersham Biosciences.
9. Laemmli buffer 3X: 150 mM Tris-HCl, pH 6.8; 6% SDS; 30% glycerol; 0.1% bromophenol blue (stored at room temperature).
Speed-Vacuum concentrator. Slab gel dryer (Hoefer).
Proteins are fixed in fixation buffer (5:1:5: 5 volumes of water, 5 volumes of ethanol, 1 volume of acetic acid) and stained with Coomassie brilliant blue R250 (Sigma) at 2.5 g/l of fixation buffer. Destain buffer is the same solution without Coomassie blue.
4. [14C]NEM-labeled extracts are exposed to general purpose Phosphor screens (Amersham Biosciences), and analyzed using a Phosphorimager with the Image Quant analysis software.
5. Fluorescent extracts are analyzed with an Odyssey infrared imaging system (Li-Cor Biosciences).
2.6. Detection of 1.
Labeled Protein Thiols 2.
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