I am trying to evaluate BSA denaturation state with GdnHCl by fluorescence emission. I'm using 0,25mg/ml BSA with a neutral density filter of OD= 1,0 and excitation wavelength 285 nm. My method is to titrate from 0 to 3M GdnHCl to obtain a sinusoidal denaturation curve, but when I tried 3M GdnHCl to see if I would get a saturated signal, fortunately I didn't. But the native protein (on an intensity scale of 1-1000) showed intensity of 480 and 3M denatured solution had an intensity of 485 (only +5, which is about 1%). Can anyone see what might be the problem AND: theoretically BSA uncoils when denatured, so tryptophans should fluoresce more. However, some articles say they suffer quenching from solution molecules. So what is the answer?
May be you should look to protein fluorescence maximum wavelength, not to its intensity? P.S. Does your GdnHCl has significant absorption at 285 nm? Than could influence the results.
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