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-   -   Problems with Agarose EMSA - Please Help! (http://www.molecularstation.com/forum/electrophoretic-mobility-shift-assay-forum/85831-problems-agarose-emsa-please-help.html)

evilhamster 06-02-2012 02:29 PM

Problems with Agarose EMSA - Please Help!
 
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Hi all,

I've been recently given the tast of identifying the affinity of my protein towards a synthetic oligo 19bp long. Previous work on the protein suggests an EMSA on a 2% agarose gel, run at 70V for 40min. Ethidium bromide was added to the agarose gel before cooling to resolve the bands quicker.

I initially observed bands in the protein+DNA lane, however as we increased the concentration of the protein, the DNA band below didn't seem to get any feint.

So I ran a lane with just protein (~100uM), and got this consistent band, heavier than the DNA control but consistent with the DNA+Protein lanes. We thought it might be a DNA contaminant so we ran it through a heparin column, but the band was still present.

We tried running mutants of the protein with which the crystal structure has been resolved with no DNA contaminants, and the same band was observed, but with varying thicknesses. (See image)

Denaturing the protein with Guanidinium-HCl, which I thought would liberate the 'mystery band' only caused a pretty much insoluble complex which wouldn't migrate through the gel.

I honestly don't know what's causing the band: since the gel already has EtBr I would assume that anything that fluoresces is DNA. :sad:

Where is that band shift? D:


Please Help,
evilhamster

mmorgan 08-16-2012 04:07 PM

Re: Problems with Agarose EMSA - Please Help!
 
Gel shifts won't work with EtBr in the gel, that's probably why you have no band shift. EtBr intercalates the DNA and disrupts Protein-DNA interactions. You need to stain with EtBr after you run the gel. Alternatively you could label the probe with 32-P so that you only visualize the probe.

As to your mystery band, I don't know what that is, but it certainly seems to be associated with your protein preps. Are you adding 100uM protein to your binding reactions?!?! That is TONS of protein. Most transcription factors will saturate the probe at 100nM (1,000 times less than you're adding).

I am a little suspicious of agarose EMSAs. I got very odd results when I did these with a known DNA binding protein. In the end I went back to the classic method of polyacrylamide gels and 32-P labelled probes and everything worked great.

Another problem with doing agarose EMSAs with EtBr staining is that you need to add tons of DNA to visualize the band. This means that you can't do the EMSA in a quantitative way. With 32-P labelled probes you can add a tiny amount of DNA (1 femtomole) and then titrate the protein to determine the Kd.


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