Reliant on different analysis reasons, 1% FBS could possibly be contained in the glucose-free moderate to lessen cell death count following the relatively harsh hypoxic experimental circumstances. 9Hypoxic treatment of myocytes may also be completed via formation of the layer of oil in the top of glucose-free medium. availability and advancement of particular antibodies against the 70 kDa subunit from the complicated II (2, 8), 3-nitrotyrosine, and GSH, provides facilitated the study of oxidative modifications of the complex II in myocytes and myocardium using immunefluorescence microscopy and immunoprecipitation techniques. Through the approach, we are able to detect enhanced protein nitration of complex II under the conditions of hypoxia/reoxygenation. 2. Materials 2.1. Gear Olympus fluorescence microscopy (Olympus American Co., model IX-71, Center Valley, PA) with 40 objective (Olympus UApo/340). Nano liquid chromatography tandem mass spectrometric analysis is performed on a Thermo-Fisher LTQ mass spectrometer equipped with a nanospray source operated in positive ion mode. The LC system is usually a Dionex Ultimate 3000 Thermo Fisher. A 5-cm 75 m ID BioBasic C18 column packed directly in the nanospray tip is used for p38-α MAPK-IN-1 chromatographic separation. 2.2. General Reagents and Supplies Peroxynitrite (OONO?) is purchased from Cayman Chemical (Ann p38-α MAPK-IN-1 Arbor, MI). BCNU (Carmustine, a potent glutathione reductase inhibitor), GSH, NADH, sodium dithionite and all chemicals are purchased from Sigma-Aldrich unless indicated otherwise. The monoclonal antibodies against 3-nitrotyrosine or GSH are purchased from Upstate Biotechnology, Inc. (Lake Placid, NY) and ViroGen Corp. (Watertown, MA) respectively. The polyclonal antibodies against the 70 kDa subunit (AbGSC90) of complex II are generated in house (2, 8). Secondary antibodies conjugated with fluorochrome are from Invitrogen, Life Technologies Corporation (Grand p38-α MAPK-IN-1 Island, NY). Gelatin/fibronectin pre-coating agent: autoclave 0.02% gelatin (0.1 g gelatin into 500 mL water), dilute 500 L of fibronectin (received as 0.1% solution) in 99.5 mL of 0.02% gelatin to make the gelatin/fibronectin pre-coating agent, and stored at 4 C up to 2 weeks. Supplemented Claycomb medium p38-α MAPK-IN-1 (complete growth medium of HL-1 myocytes): Claycomb medium, 10% fetal bovine serum, 100 U/mL-100 g/mL penicillin-streptomycin, 2 mM L-glutamine, and 100 M norepinephrine (Sigma-Aldrich, St. Louise, MO), and store at 4 C up to 2 weeks (9). Hanks balanced salt solution Csf3 (HBSS): sodium chloride (8 g), potassium chloride (400 mg), potassium phosphate monobasic (KH2PO4, 60 mg), glucose (1 g), sodium phosphate dibasic (Na2HPO4, 47.9 mg), sodium bicarbonate (350 mg), to a final volume of 1 L with water, and store at 4 C. 2.3. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) NuPAGE? Novex? 4C12% Bis-Tris pre-cast mini gels 1.0 mm is purchased from Invitrogen (Carlsbad, CA). Sample buffer (4): Tris base (0.682 p38-α MAPK-IN-1 g), Tris-HCl (0.666 g), glycerol (4 g), SDS (0.8 g), EDTA (6 mg), Serva Blue G250 (0.75 mL of 1% solution), Phenol Red (0.25 ml of 1% solution) dissolved in 10 mL ultrapure water. The pH of 1 1 sample buffer should be ~ 8.5. Do not use acid or base to adjust pH. MES SDS Running buffer (20): MES (2-(N-morpholino) ethane sulfonic acid, 97.6 g, 1M), Tris Base (60.6 g, 1M), SDS (10.0 g), EDTA (3.0 g), dissolved in 500 mL of distilled water. Do not adjust pH with base. The pH of 1 1 running buffer should be ~ 7.3. Pre-stained full-range Rainbow? molecular weight markers are purchased from GE Healthcare (Fairfield, CT). 2.4. Western Blotting for Protein S-glutathionylation and Protein Nitration Transfer buffer (20): Bicine (500 mM), Bis-Tris (500 mM), EDTA (20.5 mM), Chlorobutanol (1 mM) prepared with ultrapure water. 1 buffer should be pH 7.2. GE Nitrocellulose Pure Transfer Membranes (GE Healthcare), and TRANS-BLOT? paper, 15 20 cm (BioRad). Tris-buffered saline with Tween-20 (TTBS): 25 mM Tris-HCl, pH 7.4, 137 mM NaCl, 0.1% Tween-20. Blocking buffer: 5% (w/v) non-fat dry milk in TTBS. Antibody dilution buffer: TTBS supplemented with 5% non-fat dry milk. Primary antibody: anti-3-nitrptyrosine polyclonal antibody, anti-GSH monoclonal antibody, and AbGSC90. Secondary antibody: Amersham ECL?-horseradish peroxidase linked anti-rabbit IgG antibody (GE Healthcare). Amersham Enhanced Chemiluminescent ECL? Western Blotting Detection Reagents (GE Healthcare). Amersham Hyperfilm ECL? (8 10 inches) (GE Healthcare). 2.5. Mass Spectrometry to identify the specific site of oxidative modification Staining solution: 0.25% Coomassie Brilliant Blue R-250 in solution containing 45.4% (v/v) methanol and 9.2% (v/v) acetic acid. Fixing solution: solution made up of 40% (v/v) methanol and 10% (v/v) acetic acid. De-staining solution: solution made up of 0.5% (v/v) ethanol and 0.75% (v/v) acetic acid. Washing solution: solution made up of 50% (v/v) methanol and 10% (v/v) acetic acid, Other buffer/reagents used for the digestion: 100 mM ammonium bicarbonate.