FP signal is fine but fluorescence intensity decreases upon adding protein in my FP assay?

11 November 2016 0 5K Report

Hi,

I am new to FP assays. In the initial expt. to check binding, I am using FITC-peptide (50mM) and titrating it against 0-10uM of protein. I tried 4 conditions:

a. 20mM Tris pH 7.4, 0mM NaCl

b. 20mM Tris pH 7.4, 150mM NaCl

c. 20mM Hepes pH 7.5, 0mM NaCl

d. 20mM Hepes pH 7.5, 150mM NaCl

Though the polarization signals are similar in Tris and Hepes buffers, the fluorescence intensity decreases only in case of 20mM Hepes pH 7.5, 0mM NaCl upon the addition of protein sample, when compared to the blank (containing only peptide). Why is that so?

Is Hepes buffer not a good idea to work with in this case?

Thank you.

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