Hi,
I am doing a competition assay: {protein] is 250 nM and [fluorophore} is 30 nM (the Kd of the interaction is 250 nM). The highest concentration of inhibitor is 10 uM and I'm doing a 1 in 2 serial dilution of the inhibitor. The total volume is 20 uL in a 384 well microplate.
Buffer: 35 mM HEPES pH 7.4, 1 mM DTT, 0.007% Nonidet-P40.
At the highest concentration of inhibitor, there is negative polarization (e.g. -11 mP). I don't understand how that is possible. I initially though that I needed glycerol to stabilize the system so I went up to 10% glycerol and I was still seeing the same effect. I increased the [fluorophore] to 300 nM and had to then increase the [PCNA] to 800 nM to get the same result but now I'm far away from the Kd of the interaction so the IC50 isn't very reliable. I have tried this is a 96-well microplate format and still get the same result.
If anyone has some thoughts that would be greatly appreciated!
Check your inhibitor for fluorescence or fluorescence quenching. If you collected the parallel and perpendicular intensity data, calculate the total intensity (parallel + 2xperpendicular) without adding the protein.
If the inhibitor causes an increased fluorescence intensity, you can measure the amount and subtract it separately for parallel and perpendicular measurements before calculating polarization, as long as the amount isn't too much higher than the fluorophore's fluorescence.
If the inhibitor causes fluorescence quenching, you can still correct for it using a ratiometric method, again separately for parallel and perpendicular intensities.
Please see this paper for more details.
http://www.ncbi.nlm.nih.gov/pubmed/19643982
It is agreeable that negative polarization is impossible in mathematical sense . For displacement curve to be mathematically correct one can adjust to the point with lowest polarisation value (highest inhibitor concentration in this case). If this results in an oddly-shaped curve, then there is additional intensity factor coming from inhibitor as mentioned earlier. Subtracting this effect is probably most rational.
Actually, fluorescence polarization values can be negative even without interference of any kind. True polarization values can range from -0.33 to +0.5.
In many instances, the true polarization values are not measured because the G-factor is not accounted for, and only relative polarization is measured because only the change in polarization is of interest. In such cases, if it is desired to keep all values positive, then the instrument settings can be adjusted to make sure the lowest polarization measurement is positive.
Please keep in mind: the polarization result also depends on solvent viscosity, and adding glycerol changes this tremendously!
I'm performing FP dna binding assa using a Infinite 200 tecan, at the moment we can't calculate the proper G factor and Iìm getting negative values both for Polarization and anisotropy. there Is a way to bypass this problem untill I will have all the necessary reagents to measure it?
How i can set up a FP binding experiment if I know that my protein has multiple binding mode with the substre? (like forming 2 different complex)
Thank for the help
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I probably get negative values due to the not estimated Gfactor so the parallel intensities are never higher than the perpendicular (cause they are not corrected).
To adjust the polarization baseline to be positive, without entering a measured G-factor, set the G-factor to 1 and use different gain settings for the parallel and perpendicular measurements.
If the two binding modes have sufficiently different Kds, it may be apparent from the shape of the polarization versus concentration plot, if the data quality is very high and the concentrations are spaced closely enough together. Instead of fitting the curve to a simple isotherm for a single equilibrium, fit it to a double equilibrium binding equation and see if it produces a significantly better fit.
Obviously the observed polarization can also depend upon the excitation wavelength. What is the fluorophore you are using and what excitation and emission wavelengths are utilized? With fluorescein bound to BSA for example excitation at 480 nm (or generally in the visible range) can give polarizations up to about 0.4, Exciting the same solution at 325nm though can yield negative polarization, e.g., -0.20.
Thank you all for your replies.
Adam: I checked the inhibitor and there was no fluorescence or fluorescence quenching.
Daivd M Jameson: the fluorophore is 5-carboxyfluorescein. The excitation is 485-12 filter and the emission is 520 nm.
Just so I understand from the discussion, polarization values can be negative?
Can I ask - what is the highest polarization value you see before you add the inhibitor?
OK - so it seems to me that your high value is rather low. I wonder if your entire system is somehow out of calibration? If your G value is not correct that could lower all of your readings and even send them negative. I think you need to check that the instrument is reading properly and one test would be to use fluorescein in glycerol (you should see a high value of at least 440 MP) and also in alkaline buffer (you should see about 2 mP). If the reading are OK for this test then you can start to worry about your actual system. Good luck!
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