Hello everybody,

we have a problem with EMSA...

We use endlabelled biotinylated dsDNA (labelled with Pierce-Kit) an want to do EMSA with the Pierce lightShift-Kit...

There are no problem with the gel, our "simple" problem is to DETECT the biotin-dsOligos (some 30mers) on the gel, we can detect the non-labeled DNA but not the biotin-tagged version. The results are the same with the positive control of Pierce?

We can´t move on without solving the problem!!

Can anyone help us??

Thank you very much in advance!

Best wishes,

Gerhard

I am using the same kit.

I'm using the Promega In Vitro translation kit as the protein source which is also suggested by Pierce themselves - the kit did not work when I used the nuclear extraction kit - and the reticulosyte lysate itself is giving a signal with this detection system even when no biotinylated oligos are present!

Any suggestions?

Steve

I had many of the same problems... what % gel are you running? I've found with a 6% gel most of the IVT junk barely enters the gel, staying well away from my bands. of course, 6% might also be too restrictive for your complex to enter, but its worth a shot.

I also haven't been able to get my nuclear extracts to work, although in vitro trans. seems to work well for some of my probes.

Steve- remember that cells have biotin in them!

I have the same problem as well! I'm using pierce kit to end lable ssDNA probe with biotin and then anneal it. But in EMSA, I couldn't detect my probe after eletroblotting! I have tried streptavidin-HRP and avidin-HRP but none of them worked. I suspect the problem might be electroblotting process but just can't figure it out......

bullfrog, I have labelled with Pierce's 3' biotin endlabel kit thingy and gotten good emsa results...but here's the kicker, I use agarose gels and set up a southern

if you have attempted detection via multiple methods, are you using a kit(s)?