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Assay Method Information

Assay Name:  Steady-State Kinetics
Description:  Reaction mixtures containing twelve different concentrations of FAM-labeled DF1 (Supplementary Fig. 5a) substrate were prepared in reaction buffer (RB; 55 mM HEPES pH 7.5, 110 mM KCl, 80 mM MgCl2, 0.1 mg/mL bovine serum albumin, 1 mM DTT) and incubated at 37 °C for 10 min. Reactions were initiated by the addition of hFEN1-336Δ in RB. Reactions were sampled at seven time intervals between 2-20 min and quenched with excess EDTA (250 mM). The steady-state kinetic parameters of hFEN1-336Δ with DF1 were determined as above at various concentrations of 1, 2 and 4 (0, 5, 10, 50, 100, 500, 1000 nM) diluted from DMSO stock solutions as required. For each inhibitor concentration, reactions were followed in triplicate (each replicate using an independent serial dilution of enzyme) at six different concentrations of DF1 (10, 50, 100, 500, 1000, 5000 nM). Each experiment was independently conducted twice in triplicate.
Affinity data for this assay
 

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Last update November 1, 2007
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