Need help with tryptophan fluorescence assay

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thor310
thor310's picture
Need help with tryptophan fluorescence assay

I'm trying to determine Kd values for protein:RNA binding using tryptophan fluorescence.

Buffer:
10mM HEPES (pH 7.5)
100mM NaCL
4mM MgCl2
1mM TCEP

[protein] = 0.25uM; RNA is titrated in 0 - 4uM.

Instruments used:
-Perkin Elmer LS 55
-Varian Cary Eclipse

Fluoresence quenching data is corrected for the inner filter effect ad fit to a quadratic equation.

Problem:
Im measuring Kd's in the nM range for my protein:RNA complex, but when I titrate RNA into BSA or lysozyme as negative controls, I also measure nM Kds. Same goes for my protein:poly-dIdC.

This is very confusing. Why would I be measuring such tight binding amongst molecules that should have no specific binding?

I've increased [NaCl] to 250 nM (to reduce non-specific binding), conducted the experiment in 25% glycerol (to reduce collisional quenching), neither have alleviated the issue.

Can anyone provide rationale for my results and suggest experiment to separate the specific vs. non-specific interactions that may be leading to my results?

thanks.

Hatuey
Hatuey's picture
Unfortunately W fluorescence

Unfortunately W fluorescence is subject to many artifacts it could change both due to quenching but it could increase due to a change in environment or to a change in conformation, I have been there; check if the RNA alone doesnt contribute to the fluorescence bckd or produces a screen effect, or if the buffer where the RNA is does something like that. If you have FP installed in your instruments, you could try to measure W FP, sometimes it is more reliable, or label the protein with a fluorescnce dye

chen2828
chen2828's picture
hi!

hi!

how did you find which buffer do you need for w fluorescence?
i am beginning my research...

i looked in the internet, but i can't find smth.

thx for every answer!