Hi everyone, I'm measuring free thiol groups in relatively turbid protein samples using Ellman’s reagent DTNB (Buffer: 0.086M Tris, 0.09 M Glycine, 4 mM EDTA, 8M Urea, pH 8) and a double-beam spectrophotometer. And I’m wondering about the best way to handle blank correction.
Would the following approach be correct?
Also: Would it be even more accurate to do each measurement at two wavelengths (e.g. 412 nm for the DTNB reaction and 700 nm for background light scattering) and subtract the 700 nm signal from the 412 nm value?
(I'd prefer not to centrifuge the samples to remove insoluble protein fractions, because these might still contain free thiol groups, which I’d like to include in the measurement.)
I'd be very grateful for any thoughts, protocols, or papers that address this!