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| | Isothermal Titration Calorimetry (ITC) |
| Description: | Eya proteins were purified via S200 in isothermal titration calorimetry (ITC) buffer (50mM NaPO4 buffer, pH 6.5, 50 mM NaCl). Inhibitor solutions were prepared by diluting from a DMSO stock into ITC buffer. DMSO was added to the protein solution to the same concentration as the inhibitor solution (0.2%), and all solutions were degassed. ITC experiments were performed with a VP-ITC microcalorimeter (MicroCal, Inc.,GEHealthcare) at 25 °C. Due to low solubility of inhibitors in aqueous buffer, we injected Eya protein into the sample cell containing inhibitor, and the reference cell was filled with ITC buffer. After a 2-μl initial injection, 10-μl aliquots of 250 μM protein was added stepwise at 5-min intervals into the sample cell containing 20 μM inhibitor until saturation. |
| Affinity data for this assay | |
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