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Assay Method Information

Assay Name:  Isothermal Titration Calorimetry (ITC)
Description:  ITC was performed using LeuRS CP1 domain wild-type, K510E mutant, or K510A mutant against AN2690-AMP at 25 C by an ITC200 system (MicroCal, GE Healthcare). Wildtype protein and mutants were dialyzed against the titration buffer (50 mM HEPES-KOH, 30 mM KCl and 30 mM MgCl2, pH 8.0). Compound AN2690 and AMP were also prepared in the same buffer. A 60 μM protein solution was in the presence of 10 mM AMP in the calorimetric cell and titrated using 26 stepwise injections of 1.5 μL each with the compound AN2690 plus AMP. AN2690 solutions were placed in the syringe at 0.7 to 3 mM (depending on the expected affinity) along with 10 mM AMP. The heat generated after each ligand injection was measured base on the integral of the calorimetric signal. The resultant binding isotherms were analyzed by a nonlinear least-squares fit of the experimental data to a single site model using Microcal Origin 7.0 software. Experiments were duplicated and variability estimated to be 5% in the binding
Affinity data for this assay
 

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Last update November 1, 2007
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