These dCTP fluorescent nucleotides were incorporated into a DNA probe and I ran the free probe in the first lane from the left and cell lysate with the labelled probe in the second lane in 6% nondenaturing EMSA gel. I wanted to check for any DNA-protein complexes, but I got the image shown below. I used an appropriate filter for excitation and emission of the utilized fluorophore. I was expecting a band of the DNA-protein complex but nothing was shown apart from a smear on the top of the band that most likely represents the free probe. The bottom band most likely due to unincorporated fluorescent dCTP because I did not separate the unincorporated dCTPs from the incorporated ones. The reason is that Sephadex mini column purification had resulted in a dramatic loss of the material when it was tried. Does anybody have any suggestions as how I could make this technique work better and resolve any DNA-Protein complexes in the band shift. N.B., the cell lysate utilized in this experiment is known to contain the DNA-Protein complexes when it was tested with radio labelled dCTPs. I have seen in the literature that fluorescent EMSA is comparable to radioactive EMSA yet I could not see a discrete band shift- I just got a smear- which I think could be resolved had I used a higher percentage EMSA gel.
There are so many reasons for not getting shift.
You can get idea from attached paper.
In troubleshooting section, they mentioned different possibilities of failure.
I don't think there is much difference between fluorescent EMSA and Radioactive EMSA in terms of sensitive.
Have you checked buffer condition in which you are running the EMSA. Be careful with temperature fluctuation during running the gel.
I would like to know, please, whether the sensitivity of fEMSA is any near to the sensitivity of rEMSA. The reason is that I have tested the same probe previously with radioactivity and I have purify it using the G50 Sephadex and it gave good results. That is why I have used the same purification step with the fluorescent probe. I have been informed by the vendor that the fluorescent probe is comparable to the readioactive counterpart and it does not have any untoward consequences on my technique. Yet, I could not get the results I was looking for. I could see bands corresponding to the free probe and others to the unincorporated moiety, nonetheless, there is no protein-DNA complex are shown in the gel. I would like to know whether anybody has tried fEMSA using Texas Red dCTPs particularly. And whether they have obtained results for that matter.
Thank you
Best regards
Hi everyone,
I am desperately trying to get any shift with fluorescent probes. I am pretty sure the assay is working but I can't really see any decent band shift. Are we all sure fluorescent EMSA and radioactive have the same sensitivity? I was wondering how much of probe do you guys use for run? I am using 20 nM and I can see clearly the free probe but no shift. Any ideas please?
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