"super band" in my EMSA

[BlackAngel]

Hello, everyone!

I am using a purified recombinant protein to study the DNA binding.
I found out some "super bands" which migrate on top of the gel (closer to the loading well) at higher protein concentration (above apparent Kd). These bands are on top of the pr-DNA complex band and not at the same position but keep closer to the loading wells.

DNA is P32 labeled 66bp long dsDNA 5'-overhang.
Protein concentration is kept in the range of [0, 500nM]. If the apparent Kd is about 150nM, the super band showed up at around 200 nM and the original Pr-DNA band would disappear.

I suspect them as pr-DNA aggregates. But I am not sure. Could anybody give me a hint? I'd like to upload the image, but don't know how.

Thanks a million!

[andyzhoucang]

hi,blackangel!
if you wanna upload images,you can click "manage attachments" or"go advanced" ,then you can do it.
it seems like that there was the same confusion in our EMSA.
it's in my photo that a long "super band" migrated at the top of the gel!

[admin]

Hi there,
if you can upload your gel i can take a look at your supershifts and EMSA

[BlackAngel]

Thank you all for your kind help!
I am attaching my gels in the attachment.
I compared 2 proteins' binding affinities using two different DNAs. 66G1 is the 66ds DNA that bears one gap in the middle. 66-5'R is the 5' recessed dsDNA.
Please let me know your suggestions.

[admin]

Very nice gels and shifting however I am not sure I understand the conditions. Did you use any antibodies for supershifts? If not, it is possible that the proteins are binding in dimers/larger complexes? Also is this a denaturing gel or non-denaturing?

[BlackAngel]

Thank you very much for your reply.
I didn't use any antibodies. This is a non-detaturing gel. I also guess that the supershifts are caused by larger complexes. That is, at higher protein concentration, more proteins bind to the same DNA, and there is no single stoichiometry. How should I avoid the supershifts, or is it inevitable?
Thanks!

[sask]

Originally Posted by BlackAngel

Thank you very much for your reply.
I didn't use any antibodies. This is a non-detaturing gel. I also guess that the supershifts are caused by larger complexes. That is, at higher protein concentration, more proteins bind to the same DNA, and there is no single stoichiometry. How should I avoid the supershifts, or is it inevitable?
Thanks!

Hello BlackAngel,

Did you find a way to avoid the supershifts, I am trying to chase out the binding with my recombinant protein by adding the cold DNA fragment, but do not see any change in band intensity. I am using about 100ng of recominant protein and 1 fmol of 32P labeled DNA and went upto 1400x excess of cold fragment but did not see any chasing out. Do you have any suggestions.