How to use promaga invitro translated pr. for Pierce EMSA? - translated Pr. purify needed? (Jun/01/2010 )
I am working on PIERCE lightshift chemiluminescent EMSA Kit.
As the attachment pic.
I use Promega TNTŪ 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.
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?
epibio on Jun 2 2010, 08:08 AM said:
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.
That's unfortunate. It was a lot easier back in the days of radioactive probes, which was how I did all my EMSAs!