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High backround in EMSA

EMSA High background

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#1 aalsaegh01



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Posted 14 May 2012 - 04:13 PM

Hello All,

I am seeking a help or advice on my EMSA and I will try to explain what I am doing in details in the next paragraph.

I am doing EMSA to investigate the effect of a single nucleotide polymorphism (SNP) on the binding of transcription factors. As a control, I have been using biotynlated SP-1 probe (approx. 20bp) together with nuclear protein extracted from A549 cells (extracted using nucbuster kit). The procedure is done using 4% polyacrylamide gel and using LightShift Chemiluminescent EMSA Kit from Pierce. The binding reaction is done in a total of 20μl following the attached protocol. In my result, I am suffering from high background observed only in the column containing both probe and nuclear extract. This high background is not observed in only probe or only nuclear protein column, indicating that there could some non-specific binding of different proteins to that probe. In each experiment, I am using positive and negative controls using EBNA extract and biotynlated EBNA DNA and a clear band of the expected size is observed in the positive controls with no band in the negative control.

I have been trying different optimization strategies and different additives summarized in the following bullet points:
  • Using concentration gradient of SP-1 probe.
  • Using concentration gradient of A549 nuclear protein.
  • Incubating the A549 nuclear protein with poly dI.dC for 20min before adding the probe.
  • Change all reagent (nucbuster kit, LightShift Chemiluminescent EMSA Kit, Chemiluminescent Nucleic Acid Detection Module)
  • 50% Glycerol
  • 100mM MgCl2
  • NP-40
  • EDTA
  • KCl
  • NaCl
  • ZnCl 5uM
  • And all of the above
Unfortunately, none of these strategies or additives improved the result and the background still exists with all optimization steps.

I have also used other probe and give the same result!

Thanks for having the time to look into my problem and hope you will have an answer to my issue

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