Chemiluminescent EMSA-non specific binding to biotin - (Apr/17/2008 )

Hi all
I've been using the Pierce Chemiluminescent EMSA kit to do some band shifts. I am using biotin end labelled oligos (labelled with biotin dCTP) and 5-10ug of nuclear extract in binding buffer, protease inhibitors and 1ug of polydIdC. I incubate the nuclear extract +binding buffer, protease inhibitors and polydIdC +/- unlabelled oligos or antibody to my protein of interest for 10 mins at RT and then add 20fmol of my biotin oligos and incubate for a further 15-20 mins at RT. I see a broad smear of very bright bands at the top my 6% polyacrylamide gel and then lower down some fainter bands some of which are only in the oligo and nuclear extract lanes and others that are present in all lanes, including when unlabelled oligo or antibody has been included in the binding reaction. I was just wondering whether it is:
1. Normal to see a bright band/s of something binding to my biotin labelled oligos at the top of the gel?-Is this non specific binding?
2. Normal to see some bands that can't be prevented by adding 200x excess unlabelled oligo.

I'd be really grateful for any help with interpreting this!
Thanks
ClaireP

Hi Claire,

The bands that are at the top of your gel, do you think that they might correspond to protein stuck in your well. Do they occur where your well would be?? I'm having that problem anyway. The bands aren't very bright but they are quite defined.

As for bands that can't be gotten rid of my adding 200X cold probe.. They probably are non-specific bands.. I'm also having this problem. You could try decreasing the amount of protein that you are using, and add more pdI-dC.

I also think that you could increase the time of your binding reactions. I incubate on ice for 45 mins (extract, competitor, pdI-dC and binding buffer). I then add my probe and incubate for a further 60 mins. You could give it a try.

regards,

Sara

Hi Sara
I cut the wells off the gel before transfering the DNA to a positively charged membrane so its not protein stuck in the wells but just high up the gel. I'll try adding less nuclear extract and a longer incubation time and see if that helps. Apparently the bands at the top might have something to do with the composition of my binding buffer. A binding buffer that includes BSA might help alievate this. I'm going to try several different binding buffers and I'll let you know how it goes. Good luck with your EMSAs!

Hi Claire,

Yeah I'm also attempting an emsa with BSA added to the binding buffer.. I'll let you know how mine works. Ideally your binding buffer composition should have a salt concentration which is close to that of the salt concentration of your cell type.

sara