We use an assay to determine the binding kinetics of norfloxacin to a
target protein using radio-labelled norfloxacin. The assay is based on
a discontinuous time-course in which each time-point is derived by
sampling from a reaction mixture and filtration to trap bound
target-norfloxacin complex and remove unbound, radio-labelled
norfloxacin. Unfortunately the norfloxacin appears to bind to the
filter membrane we use which are Millipore GSTF -mixed cellulose esters
(nitrate/acetate)- type, 0.2uM pore size. Filtration and binding to the
membrane does not rely on precipitation of protein-norfloxacin
complexes prior to filtration nor does it rely on the binding of the
protein to the membrane filter.
Does anyone have any experience and advice using filtration-based,
discontinuous assays to determine the binding kinetics of norfloxacin
and other fluoroquinolones to a target protein.
Thanks in advance.
Chris Hoyle
------------------------------------------------------
Chris Hoyle
Department of Biochemistry and Molecular Biology
University of Leeds phone +44 0113 2333172
Leeds LS2 9JT FAX +44 0113 2333167
Great Britain e-mail BMBCKH at biovax.leeds.ac.uk
------------------------------------------------------
