| Assay Method Information | |
| | Calorimetric (ITC) Assay |
| Description: | For standard ITC analysis of ligand binding, proteins were dialyzed overnight in 25 mm Hepes (pH 7.5), 100 mm NaCl, 5 mm -mercaptoethanol, and 5% glycerol at 4 °C. Ligands (i.e. AdoMet, AdoCys, pEA, and pCho) were prepared in the same buffer. AdoMet and AdoCys concentrations were determined spectrophotometrically (A260 nm; = 15,400 m 1 cm 1) (19). ITC experiments were performed using a VP-ITC calorimeter (Microcal, Inc.). Injections (10 ul) of ligand were added to a sample solution containing protein using a computer-controlled 250-ul microsyringe at intervals of 5 6 min. |
| Affinity data for this assay | |
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