2.1. Sample Preparation for the Detection of Protein Carbonyls
1. Sample homogenization buffer (pH 7.4): 10 mM N'-(2-hydroxyethyl)piperazine-N -2-ethanesulfonic acid (HEPES), 137 mM NaCl, 4.6 mM KCl, 1.1 mM KH2PO4, 0.6 mM MgSO4, 0.5 pg/mL leupeptin (stored as an aliquot at -20°C), 0.7 pg/mL pepstatin (stored as an aliquot at -20°C), 0.5 pg/mL type II S soybean trypsin inhibitor, and 40 pg/mL PMSF in ethanol (prepare fresh).
2. DNPH Solution: 10 mM 2,4-dinitrophenyl hydrazine (Aldrich, Milwakee, WI) prepared in 2 N HCl solutions. This solution can be stored at room temperature.
3. Protein precipitation: Trichloroacetic acetic acid (100%, stored at 4°C).
4. Lipid removal wash: 1:1 (v/v) ethanol/ethyl acetate made fresh before use.
5. BCA reagent kit from Pierce (Rockford, IL).
6. Isoelectric focusing (IEF) tray (BioRad, Hercules, CA).
7. IEF Rehydration Buffer: 8 Murea, 2 Mthiourea, 2% CHAPS, 0.2% biolytes, 50 mM dithiothreitol (DTT), and bromophenol blue dissolved in deionized water made fresh before use.
1. IEF system (BioRad, Hercules, CA).
3. Paper wicks (BioRad).
4. Equilibration trays (BioRad).
5. Unstained and prestained molecular weight marker (Bio-Rad).
6. IEF rehydration buffer: 8 Murea, 2 Mthiourea, 2% CHAPS, 0.2% biolytes, 50 mM DTT, and bromophenol blue dissolved in deionized water made fresh before use.
2.3. Two-Dimensional Electrophoresis
2.4. Protein Staining
2.5. Oxyblot (Immunochemical Detection)
1. Agarose solution (BioRad).
2. Equilibration trays with lids (BioRad).
4. 1X running buffer (BioRad).
5. Power supply (BioRad).
6. DTT equilibrium buffer (pH 6.8): 50 mM Tris-HCl, 6 M urea, 1% (m/v) sodium dodecyl sulfate (SDS), 30% (v/v) glycerol, and 0.5% DTT dissolved in deionized water made fresh before use.
7. IA equilibrium buffer (pH 6.8): 50 mM Tris-HCl, 6 Murea, 1% (m/v) SDS, 30% (v/v) glycerol, and 4.5% iodoacetamide dissolved in deionized water made fresh before use.
8. Running buffer (10x) is purchased from BioRad and stored at room temperature.
9. Fixing Solution: 10% (v/v) methanol, 7% (v/v) acetic acid dissolved in deionized water and stored at room temperature.
10. SYPRO Ruby Stain: Purchased from BioRad and stored at room temperature.
1. SYPRO Ruby gel stain (BioRad).
1. Semi-dry transfer unit (BioRad, Hercules, CA).
2. Transfer Buffer: 1% (v/v) 10x running buffer, (no SDS)10% (v/ v) methanol diluted with de-ionized water and stored at 4°C.
3. Wash blot/ Phosphate Buffered Saline with Tween (PBST): 0.01% (w/v) sodium azide and 0.2% (v/v) Tween 20 dissolved in phosphate buffered saline (PBS) stored at room temperature.
4. Blocking Buffer: 2% bovine serum albumin (BSA) in PBST made fresh before use.
5. Primary Antibody Solution: Anti-dinitrophenyl hydrazone (anti-DNPH) antibody (Chemicon International, Temecula, CA), diluted in PBST (1:100) directly before use.
6. Secondary Antibody Solution: Anti-rabbit conjugated to alkaline phoshatase antibody (Sigma Aldrich, St Louis, MO) diluted in PBST (1:3,000) directly before use.
7. Developing Solution: SigmaFast tablet [5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT)] (Sigma Aldrich, St. Louis, MO) dissolved in 10 ml DI water, prepared fresh.
8. Nitrocellulose membrane and filter papers (BioRad).
2.6. Image Analysis 1. UV transilluminator (Molecular Dynamics, Sunnyvale, CA).
2. Adobe Photoshop on a Microtek Scanmaker 4900.
3. PDQuest image analysis software (BioRad, Hercules, CA).
Was this article helpful?