反应详情

Incubation tubes, in triplicate, receive 100 μl of [3H]NECA (94 nM in the assay), 100 μl of 1 μM cyclohexyl-adenosine (CHA), 100 μl of 100 mM MgCl2, 100 μl of 1 IU/ml adenosine deaminase, 100 μl of test compounds at various concentrations over the range of 10-10M to 10-4M diluted with assay buffer (50 mM Tris-HCl, pH 7.7) and 0.2 μl of membrane suspension (5 mg wet weight), in a final volume of 1 ml of 50 mM Tris-HCl, pH 7.7. Incubations are carried out at 25° C. for 60 minutes. Each tube is filtered through GF/B glass fiber filters using a vacuum. The filters are rinsed two times with 5 ml of the ice-cold buffer. The membranes on the filters are transferred to scintillation vials to which 8 ml of Omnifluor with 5% Protosol is added. The filters are counted by liquid scintillation spectrometry.

1. concentration/ml adenosine deaminase, 100 μl of test compounds at various concentrations over the range of 10-10M to 10-4M
2. additiondiluted with assay buffer (50 mM Tris-HCl, pH 7.7) and 0.2 μl of membrane suspension (5 mg wet weight)
3. filtrationEach tube is filtered through GF/B glass fiber filters
4. washThe filters are rinsed two times with 5 ml of the ice-cold buffer
5. additionis added