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Assay Method Information

Assay Name:  Isothermal Titration Calorimetry (ITC)
Description:  Experiments were performed on a MicroCal iTC200 titration calorimeter (GE Healthcare). Wild-type IPK1 was purified and dialyzed into ITC buffer containing 50 mM HEPES (pH 7.5), 6 mM MgCl2, 150 mM NaCl, and 1 mM tris(2-carboxyethyl)phosphine (pH 7.0). After protein dialysis was complete, dialysis buffer was used to dissolve the ligands, IP, and AMP-PNP (Jena Bioscience). Titration experiments were performed at 25 °C with 100 uM IPK1 and1mM AMP-PNP in the cell and 1-2 mM IP in the syringe to ensure a final IP:IPK1 molar ratio of at least 2:1. Titration experiments were performed at least twice for each IP, and one set was chosen to represent data.
Affinity data for this assay
 

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Last update November 1, 2007
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