I have performed fluorescence studies to understand the binding of ligands to my protein. After measuring the fluorescence study and plotting F0/F vs [Q], I found that my plot is linearly decreasing and has a negative slope. From urea denaturation studies of my protein, I had found that my protein shows increasing intensity with increasing urea concentration. How do I find the Stein Volmer constant using F0/F=1+KsV? Can the formulae be modified or can the Ksv constant be taken without the negative sign as it a constant?
Dear Santanu Sasidharan , Are you sure you plotted F0/F as a function of Quencher's concentration?. I assume F0 is the initial fluorescence response (absence of the quencher) and F is the fluorescence response with the quencher. from what I'm seeing here, once you increase your quencher concentration the F0/F value needs to be increased. In logically we have to observe a decrease in the fluorescence once we start adding the fluorescence quencher to the solution.
Chathura S. Abeywickrama Thank you for the reply. I did plot it the way you mentioned. but, I didn't get decrease in intensity rather increase in intensity. For my protein, I had observed the same in urea denaturation too where the intensity increased with increasing urea conc.
Hi Santanu Sasidharan , can you show me a snap shot of the plot you have?
Thanks
I get a plot as seen in this paper. They have used a negative slope formula. Can i use the same?
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