| Assay Method Information | |
| | KHK Inhibition Assay |
| Description: | Determination of Human and Rat recombinant KHK-A and KHK-C isozyme IC50 ValuesAn assay reagent cocktail was prepared by combining NADH, water, TEA, KCl, MgCl2, PEP, ATP, DTT, coupling enzymes (pyruvate kinase and lactate dehydrogenase, LDH) to final concentrations as shown in Table B.TABLE BReagent Final ConcentrationNADH 300 μMWater —TEA 33 mMKCl 100 mMMgCl2 6 mMPEP 1.33 mMATP 0.1 mMDTT 12 mMPyruvate kinase 1.0 U/mLLDH 1.0 U/mL[0367]To this was added the relevant KHK isozyme to a final concentration of 6 nM. Aliquots of each inhibitor compound were diluted via 5-fold serial dilutions to produce final concentrations ranging from 1000 nM to 0.064 nM. The inhibitor aliquots were added to the assay reagent cocktail containing KHK with fructose (at a concentration of 2 mM) in a 96-well plate. The absorbance at 340 nm was measured via spectrophotometry and inhibition was analyzed using non-linear regression. |
| Affinity data for this assay | |
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