As you are probably aware, the NFkB family is quite heterogeneous--lots of investigators running EMSAs for NFkB see multiple shifted bands that may vary depending upon cell type, nature of the labeled oligo (i.e. is it a "consensus" NFkB binding sequence or is it a specific sequence from a promoter of a particular NFkB-responsive gene--say VCAM-1) and physiologic state of the cells. The fact that you can compete out both bands with cold oligo indicates that you've got at least two types of complexes formed when you incubate your labeled oligo with your nuclear extract. A useful method to use to sort out what they may represent is to do supershift analysis of the bands using specific antibodies to say, p50, p52 or p65 subunits. There are other antibodies available to numerous members of the NFkB family--e.g. Abs to the Rel family; by selecting a range of Abs for supershift analysis, you might be able to dissect out the nature of the bands you see--e.g. one band may represent p50-p50 homodimers while the other one could be p50-p65 heterodimers. On the other hand, I remember one set of studies I did in endothelial cells where I consistently saw a nonspecific band (i.e. present in both control and TNF-treated cells) that I couldn't get to supershift with anything I had on hand. There are so many transcription factors floating around and some of them may stick to oligos relatively non-specifically, depending on the conditions of the assay. But try doing some supershift studies. That may provide some insight into what could be going on in your system. Good luck.
Thank you for your useful reply.
I did supershift analysis with anti NF-KB p65 and anti p50.
with p65 the top band has gone. with p50 the top band has become stronger !. but in both of them and the control one band didn't change !
so can I say that band(present in control and TNF-a treated cells) is nonspecific band ?
I'm not clear on what you mean when you say the top band is "gone" when you do a p65 uspershift--do you mean it is shifted upwards in mobility (i.e. slower migrating), or does it disappear entirely? If it has disappeared entirely, then I would worry that maybe you didn't run the gel long enough to get supershifted complexes on to the gel. I don't know why one band would become stronger when one has tried to do a supershift--I would expect that if a particular band represented p50, then doing a supershift should cause it to become entirely or partially shifted up on the gel, in which case the main band (i.e. without supershift) should decrease in intensity in proportion to how much of it was supershifted. So the one band getting stronger is puzzling to me--unless supershift of one of the components by happenstance shifts it up into another band. And yes, if you see the same band under both control and TNF-treated conditions, then it may still represent NFkB, but with respect to whatever it is TNF is doing, that band could be considered non-specific. I hope this helps a little.
Thanks for the help.
yes , the band with anti p65 did not disappeared entirly but I couldn't see the supershift band so I will try to run the gel long enough next time .
and it is a puzzl for me too the stronger band with p50 but I will try again.
hey there, i have had this strange problem with EMSA for NfKb. I am doing EMSA on nuclear protein made from mouse heart. The gels look great with two bands for NfKB which can both be competed with the cold probe. I tried to do the supershift with all the different antibodies, 65, 50, 52, relB, cRel. I only got a supershift with the p65 antibody, in that case the upper of the two bands was shifted upwards while the lower band was still there. I thought that may be the antibody for p50 is not working, got a new one, the same result. This made me believe that may be it is not there. Just to be sure that antibody for p50 was working i used a cell line, treated it with TNF and made nuclear extracts. I did gel shift for NfKb and got nice gels, this time only one band instead of two for the NfkB, when i used the antibodeis again, only p65 gave a supershift (the band was completely supershifted) but the same p50 antibode did nothing. I dont know how to explain this. Any suggestions/
I am with Ted when he said it is nonspecific band.
I tried to do the supershift as you did and I only got a supershift with the p65 antibody.
I used an inhibitor peptide p65 and the top band disappeared entirely while the lower band was still there .
I hope this will help you.
I have question related to nfkb? I submit paper recently and the reviewer was asking me about control for loading samples (he said the same control as is for western blot, the house keeping Bactin). I never saw or read somethiing like this before? Do you have any idea guys? Or the reviewer doesn't have clou about NFkb??
I use a colormetric assay to measure my protein concentration before running my EMSA samples. I've never heard about loading control for EMSA but I think you can run the same nuclear extract by WB and use
B-actin or GAPDH antibody .
I do too to know exactly how much protein to load. How about the integrity of nuclear extract (I read somewhere an assay that you can perform for this purpose) but this also is not very common?? Did you heard of this?? Thank you
Hello RH, have you find out the reason why the top band gets stronger when adding p50 antibody? I am now doing supershift assay for another predictive nuclear protein and encounter similar problem with you. Additon of a well-defined antibody dones not cause supershift band, but results in enhanced band in the same level as nuclear extract only well. Can you give me any suggestion? Thank you.
Hello, This Salman, I'm doing EMSA many times. I just wonder that why we can see multiple bands (specific, non-specific, and free Probes) in NF-kB using RAW 264.7 cells. and two bands in case of AP-1 (specific and free probes)? the oligo using is 32P.
Would u mind to answer me?? Thank you.
If an anti-bodies bind at the DNA region (or near to the DNA binding region of p65) of p65, then the suppershift band will diminished or disappear. You may order p65 anti-bodies that is against the N terminus or away from DNA binding region of p65. However, you can defend the super shift in that manner too by giving futher explaination.
Hey.. I'm doing EMSA these days for CFA-induced paw tissue, and want to check NF-kB DNA binding. but I have some problems. I used 20 ug to nuclear protein. but there was no specific binding. Would you like to give me some suggestions.