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How to use promaga invitro translated pr. for Pierce EMSA?


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#1 crazb

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Posted 01 June 2010 - 04:34 AM

Hi guys:
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. ;)

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#2 epibio

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Posted 02 June 2010 - 08:08 AM

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?

#3 crazb

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Posted 02 June 2010 - 10:36 PM

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.

#4 epibio

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Posted 03 June 2010 - 09:49 AM

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




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