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problem in chemiluminiscent EMSA PAGE running


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#1 bioindian

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Posted 04 September 2009 - 11:25 PM

dear all,
I'm using Pierce LIghtshift chemiluminiscent kit for EMSA, and getting good band shift
but when I run the DNA protein complex (includes Nuclear protein extract, labeled probe, poly dIdC, biding buffer, water and loading Dye) on 5% PAGE using 0.5xTBE in BIO-RAD mini protean assembly the dye doesn't run straight but rather gives a wave like appearance. Moreover, the samples with no probe as well as with no protein move a bit faster as compared to other ones.
Can anybody help me to troubleshoot.
regards

#2 N_L

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Posted 14 September 2009 - 10:38 PM

dear all,
I'm using Pierce LIghtshift chemiluminiscent kit for EMSA, and getting good band shift
but when I run the DNA protein complex (includes Nuclear protein extract, labeled probe, poly dIdC, biding buffer, water and loading Dye) on 5% PAGE using 0.5xTBE in BIO-RAD mini protean assembly the dye doesn't run straight but rather gives a wave like appearance. Moreover, the samples with no probe as well as with no protein move a bit faster as compared to other ones.
Can anybody help me to troubleshoot.
regards


Yes. I also got this problem with PIERCE kits and Bio-rad Mini protean system. But the interesting thing is that, this only happens when I used my own custom DNA sequences self-labeled with PIERCE biotin labeling kit. Everything was okay with PIERCE control DNA or TF probe from Panomics. I still couldn't shift with my oligo. So I couldn't bother about wave like appearance.
I used 6% gel. Run with prechilled 0.5X TBE.

Good Luck.

#3 kersin

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Posted 14 October 2009 - 07:57 PM

Ya, I have this problem too. I see the problem when I run nuclear extract reaction next to the whole cell lysate. It only happens to nuclear extract. Recently, I ran a gel which all the lanes for nuclear extract reaction, they all were straight like normal gel. I don't know whether that's because my protein concentration is higher for this time (less salt in the reaction??) or because of the electrical field is more even during the eletrophoresis.




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