We have generated very consistent FP data that shows decreasing FP with DNA/protein interactions in stead of increasing (as it should be according to the literature).
Can anyone help with how I can determine the Kd of this interaction using this data? PRISM does not like fitting a Hill-curve to it because it is inverted.
There is example data included.
Conditions:
2 nM dsDNA probe labeled 5' 6-FAM
[protein] as indicated
protein and dsDNA probe mixed and incubated at RT for 30 before measuring FP
My first guess is that the DNA is getting degraded by a trace amount of DNase contaminating the protein. If you do not have any EDTA in the buffer to bind up Mg2+ (which is required for DNase activity) try adding 1 mM.
A second guess is that there is a trace of some fluorescent material with a low polarization in your protein. Check this by doing the same titration without the DNA. If you see any background from the protein, subtract it from the parallel and perpendicular intensities of the DNA + protein titration at each protein concentration, then recalculate the polarizations.
Another observation is that your starting polarization seems very high at 20 nM protein. What was it at zero protein? Maybe the binding affinity is higher than you think and you should be using a lower range of protein concentrations.
Thank you for your quick response and thoughtful suggestions!
We do not have EDTA in our reaction buffer, we can definitely try that.
The DNA only control (not shown because of the log scale) has a comparable polarization to the samples with very low protein concentrations, around 250 mP, which made me think it wasn't basal fluorescence from the protein. Does this also suggest that DNase degradation is not occurring?
My initial thought was your third guess, which was that the binding affinity was very high. Although expanding the protein range (adding 1, 2, 4, 8, and 16 nM samples) did not give us different results. We still only saw a change in polarization between ~80-180 nM protein.
Does this also suggest that DNase degradation is not occurring?
No, DNA degradation is more likely to be a problem at high enzyme concentrations.
As Adam already pointed out, the starting value for what should be the free ligand is rather high. How large is this dsDNA? How did you calibrate the instrument (often called the G-factor), or what mP value do you observe for free fluorescein?
Alternatively, does your DNA aggregate, and is then released from the aggregate upon binding to the protein? Also, depending on the instrument, buffer, plate, etc 2nM might be near the detection limit.
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