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Assay Method Information |
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| | Enzyme Immunoassay (EIA)) |
| Description: | The assay is carried out by the following steps: 1. Inhibitor screening is performed in 100 mM Tris-HCl, pH 8.0 containing 1 mM GSH, 1 mM MgCl.sub.2, 4% inhibitor/DMSO, 40 .mu.M PGH.sub.2 and 25 ng of PGDS in a total volume of 125 .mu.L. 2. A reaction mixture containing 100 mM Tris-HCl, pH 8.0, 1 mM GSH, 1 mM MgCl.sub.2, 25 ng H-PGDS and 4% inhibitor or DMSO (uninhibited reaction) is preincubated at 25.degree. C. for 10 minutes. 3. Reactions are initiated using 5 .mu.L of PGH.sub.2 and quenched every 15 seconds for one minute. Reactions are quenched by taking 10 .mu.L of reaction mixture and adding to 490 .mu.L of 100 mM phosphate buffer containing 0.1% BSA, 400 mM NaCl, 1 mM EDTA, 20 mM FeCl.sub.2, 10% 1N HCl, and 0.01% azide to prevent the non-enzymatic formation of PGD.sub.2 from PGH.sub.2. The FeCl.sub.2 serves the purpose of reducing PGH.sub.2 to 12-HHT. (Quench buffer is kept on ice at all times). 4. Quenched samples (5 .mu.L) are further diluted 100 fold in 495 .mu.L of 100 mM. |
| Affinity data for this assay | |
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