Help with non-radioactive EMSA! - unlabeled oligo causes bands to appear instead of disappear!!! (Sep/16/2008 )

Hi everyone,

I am using the Pierce LightShift EMSA kit, and am having problems understanding my results. I normally see 1 band along with the free probe in reactions containing nuclear protein and biotinylated DNA. When I use unlabeled DNA for competition, this band gets darker and another one shows up below it (very odd!), as opposed to decreasing or eliminating bands. Has anyone out there every encountered this before, or have any suggestions? Are these bands just non-specific?

The only changes I have made to the Pierce protocol are including PMSF as a protease inhibitor, pre-incubating nuclear extract with buffer/dIdC (15m @ RT) before addition of oligo, and doubled the amount of biotinylated oligo per reaction. I tried these things because overall my signal seems somewhat weak.

I have also tried supershifting the band with antibodies directed toward the factors that SHOULD bind my oligo. Generally, the band gets lighter, but I don't see any larger bands showing up. I'd appreciate any suggestions!

Thanks,
Heidi

hey Heidi - could you please post a photo?

Hi,

Here is my latest attempt. All lanes have biotin labeled probe (60fmol).

1: no nuclear protein
2: 8ug nuclear protein
3: nuclear protein + antibody 1
4: nuclear protein + antibody 2
5: nuclear protein + antibody 3
6: nuclear protein + unlabeled DNA (8pmol)

The additional band coming up with the unlabeled DNA has happened every time I've tried this. I don't think my biotin labeling worked that great, but according to the Pierce protocol EMSA should work even if only 50% is labeled. Also, I've checked my nuclear protein on western blot and it has the proteins I'm looking for with the antibody supershifts.

Thank you so much for taking a look - I appreciate any and all suggestions! Let me know if you have any other questions.

Heidi

hmmmm......

there is no labeled DNA in lane #6? that doesn't make any sense at all? there must be labeled DNA, as there is a band of free oligo at the bottom?
please clarify for me exactly what you're putting into this lane?

also, one small thing that may help to clarify your bands - reduce your glycerol to straighten out your bands. (note the 'smile' and the smeary free olig)

Thanks for looking at my results.

Sorry, I made a mistake in the last post - there IS labeled DNA in lane 6. Exact components: 1X binding buffer, 8ug nuclear protein, 60fmol biotin-labeled oligo, poly dI/dc, 8pmol unlabeled oligo, 1mM PMSF. What I'm trying to do in this reaction is see shifted bands disappear because they are binding to unlabeled oligo (competition experiment), but instead bands are appearing where they did not in lane 2 (biotin labeled oligo + nuclear extract). Any ideas why this would happen?

I didn't add any additional glycerol, but there my nuclear extract buffer is 10%...can't really change this though I don't think.

Thanks again for your help!