| Assay Method Information | |
| | Human LDHA Enzyme + HSA Assay |
| Description: | Compounds were dissolved in DMSO and preincubated with human recombinant C-terminal His-tagged LDHA (0.070 μg/mL) for 10 min at room temperature in assay buffer consisting of 50 mM Tris (pH 7.5) and 100 mM NaCl in black walled, clear bottom, non-binding 96-well plates. Equal volumes of substrate solution containing 100 μM of pyruvate and 100 μM of NADH in assay buffer was added to each well (final concentration 0.035 μg/mL enzyme, 50 μM pyruvate, 50 μM NADH, and 1% DMSO). For human serum albumin (HSA) shift assay, the compounds were preincubated with the enzyme in assay buffer containing 20% HSA before substrate addition (final concentration 10% HSA). The reaction was monitored at 340 nm on a plate reader (Molecular Devices) in kinetic mode for 15 min. The rate of the reaction was determined by plotting absorbance vs time. |
| Affinity data for this assay | |
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