It's my first time to do FP assay. I help measure Kds of inhibitors.
I got two probes (hereafter P1 and P2, both containing FITC) and one protein sample (Pr). Kd(Pr, P1) is around 30 nM (done by me), and Kd(Pr, P2) is about 26 µM. And two inhibitors (I1 and I2)
[P1] = 25 nM and [P2] = 0.1 µM
[Pr] = 0, 80, 300 nM; [I1] = [I2] = 0, 80, 300 nM
For multiple conditions, one by one, totally 22 mixtures:
only probes [P1/P2] as background (2)
only probes and protein [80/300 nM] (4)
80 nM protein, probes [P1/P2] and inhibitor [I1/I2, each 80/300 nM] (8)
300 nM protein, probes and inhibitor (8)
Inhibitors were dissolved in DMSO, [P1]stock = 5 µM, [P2]stock = 2 µM
I could get A0s with only probes. However, I got increasing A values when increasing inhibitors' concentration. (Almost (.06)). When conducting a competitive inhibitor binding assay using FP, it showed very confusing and abnormal values, even with fluctuation. (one was .02-.06 in 10 scans)
Buffers were tried filtering with a .22-µm filter twice; I saw the value 0.00xx with the only buffer so that I can measure subsequent samples without suspecting contamination.
Therefore, is there any suggestion or avoidance so that I can circumvent this frustrating situation?
Some things I thought of are (1) insolubility of inhibitors causing light scattering, which has high polarization, (2) fluorescence of inhibitors (easily checked), (3) fluorescence quenching by inhibitors, and (4) insufficient fluorescence intensity of probes.
If you are using a fluorescence polarization microplate reader, make certain there are no bubbles in the wells. Also, include some nonionic detergent in the buffer (e.g. 0.005% Triton X-100) to keep things from sticking to polystyrene plates. Make sure the samples are thoroughly mixed.
FITC is prone to photobleaching. Be careful to be consistent with the amount of illumination of samples, and minimize it.
By the way, although it is probably not related to your problem, I noticed that you are working in the tight-binding domain with P1 (Kd = 30 nM and [P1] = 25 nM). It would be preferable to lower the P1 concentration, if feasible.
The data showed that I probably had a mix-up over inhibitor and FITC-conjugated inhibitor, since the probe just an inhibitor linked with a benzenediol and then FITC (amine-linked).
After re-conducting the experiment, they appeared fewer irregular ups and downs, just only at 80 nM proteins, at least, and therefore i can calculate the % inhibition.
I will test with more diluted probes and proteins to see whether it improves. Thank you very much!
[Following up, 1 month later]
Nothing to do with mixing-up. The solution appeared micelles after adding probes into the sample (peculiarly they united and sediment when I did reversely) proven by AUC and light scattering, that's the reason why I keep getting the shifting values. We decided to change another probe to prevent this outcome and got them done.
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