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Assay Method Information |
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| | in vitro DltA activity assay |
| Description: | The assay buffer AB contained 50 mM Hepes pH8.0, 10 mM MgCl2, 50 mM KCl, 0.012% Triton-X100, 10 mM DTT and 100 nM Myelin-basic protein. The following components were added in a white polystyrene Costar plate up to a final volume of 30 μL: 3 μL DMSO, or inhibitor dissolved in DMSO and 27 μL DltA enzyme in AB. After 30 min of pre-incubation at room temperature, 30 μL of Substrates mix in AB were added in each well to a final volume of 60 μL. This reaction mixture was then composed of DltA (produced in house from S. aureus, E. faecalis, E. faecium and S. agalactiae), 0.5 mM D-Alanine (Sigma), 0.5-5 μM ATP (Sigma) and 0.05 u/ml inorganic pyrophosphatase (Sigma) in assay buffer. After typically 1-2 hours of incubation at room temperature (conversion rate around 30%), 30 μL of the revelation mix were added to a final volume of 90 μL, including the following constituents at the respective final concentrations: 10000 u/ml luciferase (Sigma), 30 μM D-luciferin (Sigma), 100 μM N-acetylcysteamine (Aldrich). Luminescence intensity was immediately measured on a Fluostar Optima (BMG) (excitation 360 nm, emission 520 nm) and converted into inhibition percentages. For IC50 determinations (Inhibitory Concentration 50%) the inhibitor was added at 6 to 10 different concentrations and the related inhibitions were fitted to a classical Langmuir equilibrium model using XLFIT (IDBS). |
| Affinity data for this assay | |
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