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Assay Method Information

Assay Name:  Optical Titration Assay
Description:  Optical titrations to determine the binding affinity (KD values) of ligands were carried out on a Cary 60 UV−visible spectrophotometer (Varian, UK) according to a previously described procedure [McLean et al., J. Inorg. Biochem., 91:527-541]. Ligands were prepared as stock solutions typically 0.1−100 mM) in DMSO and added as 0.2 μL aliquots to cuvettes containing either a solution of CYP144A1 (∼4-8 μM) in 100 mM KPi (pH 7.0), containing 10 mM KCl, or to buffer alone. DMSO concentrations did not exceed 1% v/v of the final titration volume (1 mL), and the absorbance spectrum of CYP144A1 was not affected within this range. Spectra were recorded continuously between 800 and 250 nm at 25 C.
Affinity data for this assay
 

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Last update November 1, 2007
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