3 replies to this topic

[crazb]

Hi guys:
I am working on PIERCE lightshift chemiluminescent EMSA Kit.
As the attachment pic.
I use Promega TNT® Quick Coupled Transcription/Translation Systems to get my Protein truncation "A" from the literature sequence.(Positative control Luciferase translation was so good) .But when I put 2ul of my translated product together with NE into EMSA systerm, finally the membrane looks so dirty on the top in Lane 3. Lane 1 with NE only is ok.I think it is not the Pr "A" shift because Lane 2 was Luciferase product control with the same pattern.
How to avoid the contaminate?Should I purify the translated product?But Promega Kit said the product can be used dirrectly in EMSA assay.Or I put too much product in EMSA assay? Has anyone has the same problem?I will be so appreciated if anyone could give me some suggestion.

Attached Files

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[epibio]

It looks like you have too much protein. The free probe (unbound DNA) should be in large excess; try titrating the amount of extract that you add to the EMSA reactions. Also, I assume you're using something like poly(dI-dC) to soak up nonspecific binding proteins?

[crazb]

epibio, on Jun 2 2010, 08:08 AM, said:

It looks like you have too much protein. The free probe (unbound DNA) should be in large excess; try titrating the amount of extract that you add to the EMSA reactions. Also, I assume you're using something like poly(dI-dC) to soak up nonspecific binding proteins?

Thanks a lot for your reply!
It does have too much protein, I just ask the Promega service, they told me their invitro translation systerm has 100KD endogenetic Biotin-Pr.
So we can not use Promega invitro translation and Pierce biotin gelshift together.

[epibio]

That's unfortunate. It was a lot easier back in the days of radioactive probes, which was how I did all my EMSAs!