Hi All,
I am currently working on a protein-miRNA binding assay. I am using FP as my assay system to see if the binding happens. Previous publications show the protein binds to the miRNA and my data shows up the same result too.
But the problem is, higher the concentration of my protein is higher the readout should be. My highest concentration of protein is 30uM which is the original purified product, and my miRNA probe has the best readout when I use 1nM concentration.
So I fixed the probe concentration as 1nM and did the titration of my protein (by using protein purification buffer) from 1nM to 30uM. The result shows as the protein concentration goes higher, the readout is higher too, but not reach the flat yet (just an exponential line).
Since my original stock concentration is 30uM and it seems that I need higher concentration, I used an Amicon 0.5mL ultracentrifuge filter to concentrate my protein. After the centrifuge, I checked the protein concentration with the A280 Nano-drop machine, and it showed my protein concentration has increased 12-fold. However, when I performed my FP assay with a higher concentration (more than 30uM), the FP readout dropped to the baseline. I tried to modify the centrifuge process and it doesn't work (which costs me a lot of samples and the values are still around the baseline)
Most of the published FP need to have S curve to show the IC50 but I just cannot get the S curve, any suggestions? Is there anything wrong with my centrifuge process?
Thank you so much!
With such high protein concentrations, there is a risk that the protein alone is contributing to the increase in FP due to scattered light. Try doing titrations side by side with and without miRNA. Subtract the individual parallel and perpendicular fluorescence intensity of the protein alone from that of the miRNA+protein at each protein concentration to remove the contribution from the protein. Then use the corrected intensities to calculate the polarization or anisotropy. You may find that with the scattering from the protein removed, you can see the binding signal. Also, it may be worth centrifuging the protein very well before doing the titration to remove any aggregates that scatter light the most.
Adam B Shapiro Hi!
Thank you for your suggestions, but I have already got the binding signal. Let's say my baseline FP readout with just free miRNA probe is 80. I got the binding event signal up to 180 and the competition with no label probe is also making the readout decreasing. However, as I go up to higher than 30uM (after Amicon concentration) the protein concentration goes higher but the FP readout drop to 80. So the whole picture is like this:
Fixed Probe concentration at 1nM
Protein Concentration(uM): 0.1 0.5 1 5 10 20 30 40 50
FP readout: 100 115 130 145 162 175 180 80 79
The data after 30uM doesn't make any sense to me. I couldn't get a good flat area so that's why I tried to raise the protein concentration.
I am trying to put more points before 30uM to see if I can get similar readouts to get the flat area under 30uM.
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