Hello,
I am following a paper's protocol for an FP assay for my protein to try to establish an FP assay for testing small molecules.
I am starting with a protein titration. When I conduct it, the values of polarization are very low, compared to what was in the paper. As I increase the concentration of my protein, I see an almost linear increase in the polarization but it looks like a plateau is never reached. All values of polarization is small (below 100), but I do see an increase. I have increased the protein concentration to 4.5 uM, with the suggested concentration used in the paper being 3 uM.
The buffer is 50 mM borate buffer, pH = 7.5
The probe is a 6-FAM probe attached to an oligonucleotide
Plate used is a black, NBS plate
Thank you in advance for any tips and suggestions!
We add approximately 0.1% tween 20 for the Fp experiment to remove some non-specific interaction. You can add it to your protein buffer and try to see if that helps. The extent of polarization depends on the relative size difference between your labeled ligand and the protein. If they are comparable in size then you might not get a very high polarization value.
Go through this paper to know further details - Article Fluorescence Polarization (FP) Assays for Monitoring Peptide...
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