| Assay Method Information | |
| | Luminometric Assay |
| Description: | The reaction mixture contained 50 mM tris acetate (Merck, #1.08382.1000), pH 7.5, 250 μM acetyl coenzyme A (Sigma, #A2181),16 mM NaHCO3 (Merck, #1.06329.0500), 0.9 mg/ml bovine serum albumin (Sigma, #A8806), 25 μM ATP (Sigma, #A7699), 1.1 μM β-mercaptoethanol (Roth, #4227,1), 4.3 mM magnesium acetate ((Merck, #1.05819.0250), 10 mM sodium citrate (Merck, #1.06448.0500) and 200-400 ng of the ACC preparations. The mixture also contained a test compound. The test compounds were each dissolved in 10 mM DMSO and were assayed at final concentrations of 0 μM, 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM and 100 μM. The final concentration of the DMSO in the assay was 0.1% (v/v). The reaction was started by adding ACC and was carried out at 37° C. for 90 min. The reaction was then stopped by adding 150 μl of 0.5 M tris acetate. 10 μl of the mixture were removed and mixed with 90 μl of ATP monitoring reagent (Cambrex, #LT27-002). After incubation for 10 min, the luminescence was measured in a luminometer (Tecan Genios Pro). |
| Affinity data for this assay | |
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