I am performing inhibition assay of a protein by using substrate containing flourecent group modification. The inhibitory substances i am using are coloured molecules and extracts. In most of them i am getting high flourecence (close to sample) even in control containing only substrate and inhibitory molecule (without substrate). is it poosible that colour of inhibtory compound can interfere with flourecence. or what could be poosible reason of that and how can that be eliminated?
Normally, the "colorful" compounds should not interfere with the fluorescence-based assay unless these compounds are intrinsically fluorescent. Another reason for the high background fluorescent signal in your case might be the low solubility of your samples in the test buffer. If a compound/extract is partially precipitated during the assay, it might give a jump of the fluorescence signal. You can try to reduce the concentration of the test compounds in your assay. Alternatively, you can increase the concentration of DMSO in your assay (1-2%). Also, you can increase the concentration of the enzyme, which should faster and more efficiently cleave the fluorogenic substrate. In this case, the intrinsic fluorescence of the test compound should be negligible.
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