2.4. Western Blotting 1
S-Nitrosoglutathione (GSNO) is dissolved in water at 10 mM and stored in aliquots at -20°C. GSNO decomposes rapidly in solution (approx. 5% per hour in water at room temperature). To avoid faster decomposition the dissolved GSNO should be protected from light and from contact with transition metals, especially Cu(I). PD10 columns from Amersham Bioscience, (Freiburg) and Micro Bio-spin P6 columns from Biorad to get rid of low molecular weight compounds (<6,000 kDa).
2 M Methyl methanethiosulfonate (MMTS) in dimethylfor-mamide.
50 mM ascorbate in water; stored in aliquots at -20°C.
N-[6-(biotinamido)hexyl]-3'-(2'-pyridyldithion)propion amide (Biotin-HPDP) from Perbio (Bonn) dissolved in dimethylformamide at a concentration of 4 mM and stored at -20°C.
HENS buffer: 25 mM HEPES-NaOH, pH 7.7; 1 mM EDTA; 0.1 mM neocuproine; and 1% sodium dodecyl sulfate (SDS).
Separation buffer (4X): 1.5 M Tris-HCl, pH 8.7; 0.4% SDS.
Stacking buffer (4X): 0.5 M Tris-HCl, pH 6.8; 0.4% SDS.
Ten percent ammonium persulfate (APS) in water. Store aliquots at -20°C or use always freshly prepared solutions of ammonium persulfate.
N,N,N,W-Tetramethyl-ethylenediamine (TEMED) (Serva).
Prestained molecular weight markers (Fermentas).
Thirty percent polyacrylamide solution containing 0,8% bisacrylamide.
SDS-PAGE sample buffer: 50 mM Tris-HCl, pH 6.8; 2% (w/v) SDS; 20% (v/v) glycerol; and 0.01% (w/v) bromphenol blue. Store at room temperature. If proteins should be separated under reduced conditions add 2 mM dithiothrei-tol (DTT) just before use.
Running buffer: 25 mM Tris , 0.2 M glycine, 0.1% (w/v) SDS.
Towbin Transfer buffer: 25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS, 20% (v/v) methanol. Store transfer buffer at 4°C and add methanol just before use.
PVDF-membrane (0.2 pm) was obtained from Pall (Portsmouth, UK).
3. Nitrocellulose membrane (0.2 pm) was purchased from Schleicher & Schuell (Dassel, Germany).
4. Tris-buffered saline (TBS): 10X stock solution—100 mM Tris-HCl, pH 7.4; 1.5 M NaCl; and 10 mM MgCl2. Dilute 100 mL with 900 mL of water for use.
5. Tris-buffered saline with Tween (TBS-T): 0.5% (v/v) Tween 20 in TBS.
6. Blocking buffer: TBS-T supplemented with 1% (w/v) nonfat dry milk and 1% (w/v) BSA.
7. Mouse anti-biotin antibody coupled with alkaline phosphatase (Invitrogen).
8. A solution of 10% nitroblue tetrazolium chloride (NBT) (Roth) is prepared in 70% dimethylformamide.
9. A solution of 5% 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Roth) is prepared in water.
10. Gel Blotting Paper GB002 from Whatman (Schleicher & Schuell).
2.5. Purification of 1. Empty columns (BioRad, Munich).
Biotinylatedmterns 2. Immobilized Neutravidin (Perbio, Bonn).
3. Neutralisation buffer: 20 mMHEPES-NaOH, pH 7.7; 100 mM NaCl; 1 mM EDTA; and 0.5% (v/v) Triton X-100.
4. Washing buffer: 20 mM HEPES-NaOH, pH 7.7; 600 mM NaCl, 1 mM EDTA; 0.5% (v/v) Triton X-100.
5. Elution buffer: 20 mM HEPES-NaOH, pH 7.7; 100 mM NaCl, 1 mM EDTA, 100 mM ß-mercoptoethanol (add ß-mercoptoethanol just before use).
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