I am trying to do EMSAs and I am using a consensus and mutant oligo specific for my protein which I ordered from Santa Cruz and then a custom biotin labeled oligo (I have annealed sense and antisense). I cannot compete out any of the bands with self. In addition, I see the same banding pattern for all three when added with nuclear extract and then again all three show a supershift when antibody for my protein of interest is added but not when a nonspecific antibody is included. So I am guessing that the banding pattern I am seeing is nonspecific, however, I can't seem to get rid of it.
The protein I am looking at is HIF1a and I am using samples treated with 1%O2 in which I can detect HIF1a protein using WB. For samples for the EMSA I am using a NE-PER kit. The consensus and mutant sequences from Santa Cruz are also for HIF1a.
Any help or suggestions is greatly appreciated.
If competition is not working, I would try the following:
-Reduce the amount of protein to the minimum where you still see a probe shift. Specially for nuclear extracts, the effective amount of your DNA binding protein can be quite high, compared to total extracts, so make sure you are not using too much, since competition would be difficult to see even if using specific competitors.
- Reduce non-specific binding adding nonspecific DNA such as salmon sperm DNA, calf thymus DNA, or synthetic poly(dI:dC). Addition of nonspecific proteins (e.g., albumin) may also increase specific DNA–protein interactions.
- Increase the molar excess of competitor DNA. Usually 50X would work, but for some protein-DNA complexes I have seen that even 1000X excess of competitor is necessary. Try this after you have established the minimum amount of protein for observing gel retardation complexes.
Good luck!
Hi!
That problem sounds familiar. We observed a similar effect for many bacterial transcription factors. To make sure that you have a specific binding, we now always add competitor DNA to our EMSAs.
You might try either something like fragmented salmon sperm DNA or, if you want something more defined, polycytidylic acid-polyguanylic acid. The final concentration in your assay should be ~ 50ng/µl.
Hope that helps.
If competition is not working, I would try the following:
-Reduce the amount of protein to the minimum where you still see a probe shift. Specially for nuclear extracts, the effective amount of your DNA binding protein can be quite high, compared to total extracts, so make sure you are not using too much, since competition would be difficult to see even if using specific competitors.
- Reduce non-specific binding adding nonspecific DNA such as salmon sperm DNA, calf thymus DNA, or synthetic poly(dI:dC). Addition of nonspecific proteins (e.g., albumin) may also increase specific DNA–protein interactions.
- Increase the molar excess of competitor DNA. Usually 50X would work, but for some protein-DNA complexes I have seen that even 1000X excess of competitor is necessary. Try this after you have established the minimum amount of protein for observing gel retardation complexes.
Good luck!
I have been including poly di/dc in all binding reactions (following the pierce kit protocol). I started out using 6ug of protein and am now at 2ug of protein. I wasn't sure if there was a suggested minimum amount of protein.
I will also try increasing the amount of competitor. The highest I went was 500x but will try higher.
Thanks for the suggestions!
Maybe this can give you some other ideas: http://cshprotocols.cshlp.org/content/2012/1/pdb.top067470.full
Do you know if your bands are specific? Try doing a supershift by adding excess antibody after your main binding reaction has completed. Also, as others have suggested, you need to add several hundred times molar excess of unlabeled mutant ds-oligo to see the effect. Make sure you are using poly-dI-dC oligo as well to decrease non-specific signal. Since you are using biotin, make sure you are blocking sufficiently prior to adding your strepavidin-HRP. Sheared herring sperm or something similar can help with any non-specific binding as well. Good luck.
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