| Assay Method Information | |
| | Isothermal Titration Calorimetry (ITC) |
| Description: | ITC studies were carried out using a Microcal ITC200 instrument (GE Healthcare). Protein concentration was determined by spectrophotometry by measuring the absorbance at 280 nm using a theoretical molar extinction coefficient of 169,140 M−1cm−1. Ligand stock solutions were prepared in DMSO at 62.5 mM concentration. The titrations were performed at 25° C. with 7.5-100 μM E. Coli LeuRS in 50 mM HEPES, 150 mM NaCl, pH 7.5 buffer containing 1% DMSO (v/v). The protein solution in the 280 μL sample cell was titrated with the inhibitor solution (diluted to 75-3000 μM in the same buffer as the protein) using 1-2 μL injections every 120-140 seconds. All titrations were repeated at least two times. To correct for heats of dilution and mixing, the final baseline consisting of small peaks of identical size at the end of each experiment was subtracted. The experimental data were fitted to a theoretical titration curve (one site model) using MicroCal Origin 7 SR4 software. The arithmetic mean±standard deviation (SD) of K, ΔH, ΔS values from at least three experiments are shown in the following table. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |