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| | HIV Integrase Inhibitory Assay |
| Description: | The donor DNA was diluted with TE buffer to 20 nM, of which 50 μL was added to each well of streptavidin-coated black plate (manufactured by PIAS Corporation) and allowed to adsorb at 37° C. for 20 min. The plate was washed with phosphate buffer (Dulbecco's PBS, Sanko Junyaku Co., Ltd.) containing 0.1% Tween 20 and phosphate buffer. Then, an enzyme reaction mixture (70 μL), a test substance (10 μL) diluted with the enzyme reaction mixture and 0.75 μM integrase protein (10 μL) were added to each well and the mixture was reacted at 37° C. for 60 min. composition of enzyme reaction mixture: 30 mM MOPS (3-morpholinopropanesulfonic acid), 5 mM magnesium chloride, 3 mM DTT (dithiothreitol), 0.1 mg/mL BSA (bovine serum albumin), 5% glycerol, 10% DMSO (dimethyl sulfoxide), 0.01% Tween 20.Then, 25 nM target DNA (10 μL) was added, and the mixture was reacted at 37° C. for 20 min and washed with phosphate buffer containing 0.1% Tween 20 to stop the reaction. Then, 100 mU/mL peroxidase labeled anti-digoxigenin antibody solution (Roche, 100 μL) was added, and the mixture was reacted at 37° C. for 60 min, followed by washing with phosphate buffer containing 0.1% Tween 20.Then, peroxidase fluorescence substrate solution (manufactured by PIAS Corporation, 100 μL) was added, and the mixture was reacted at room temperature for 20 min to 30 min. A reaction quenching liquid (manufactured by PIAS Corporation, 100 μL) was added to discontinue the reaction, and fluorescence intensity at excitation wavelength 325 nm/fluorescence wavelength 420 nm was measured. |
| Affinity data for this assay | |
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