EMSA Supershift for STAT3 - (Aug/16/2006 )
Hi there, I need a little help for my gelshift supershift experiments. I'm looking for STAT3 and have made a lot of gelshifts, where I always see the shift for my STAT3-Oligo-Complex. But when I try to do a supershift (santa cruz antibody sc-8019-X) with a specific antibody for gelshifts, I don't get a different signal, looks just the same as before.
I have also tried to use different amounts of antibody, different antibodies (another stat3, p-stat3 tyr, p-stat3 ser) and Incubation before or after oligos. Also tried 4°C and room temperature for 30 minutes.
My binding buffer ist 10mM Tris-Hcl, pH 7,5, 50 mM NaCl, 4% Glycerol, 1mM MgCl2, 5mM EDTA, 0,5mM DTT, 1 µg poly(dI-dC) (dI-dC). I add 10 µg protein to end volume of 20 µl binding reaction. Oligos with 100.000 cpm. My Gel is 6% (19:1 Acrylamid), 0,25 TBE, 200V for 2h, prerun at 150V for 30 min at RT.
Has anybody ever done this experiment? I would be very thankful for every suggestion, how to go on.
Has your antibody been previously shown to be suitable for supershifts?
You may find the epitope is not in an appropriate location for the antibody to bind when the protein is bound to DNA.
The antibody is specifically sold for STAT3-supershift, that's why I decided to buy this. It was much more expensive, but I hoped to get a good result with it And I have seen at least one publication, where they have used this antibody for a supershift. Was a bit cloudy, but there was kind of a supershift. I get nothing...
I'm not really sure then. Do you not even get aggregation and material stuck in the well when you add the highest amounts of antibody?
A little material is stuck in every pocket, but I don't see a great difference between samples with antibody and without, Perhaps a little more with antibody, but not convincing, I think.
I use the anti-STAT3 (C20) Sc-482 for super-shifting STAT3. It works with human and mouse nuclear extracts. It's the western blotting concentration so you probably want to use Sc-482x if you're mainly doing supershifts.
All the best,
Pfitzner's Stat3 EMSAs look great. Maybe this publication helps.
Yes, those are good supershifts. It's the same ab I use too (Sc-482).
Is that your work, Jou?
I used to work on EBV so had an interest in Hodgkin's lymphoma.
Thanks, Ceri and Jou, I will try to convince Santa Cruz to give me a new antibody for free, the one you both use. I would then just like to know, how exactly you use at, so, before adding of the labeled oligos or afterwards, how long and on whicht temperature do you incubate and with which binding buffer. And which kind of gel do you use? Do you think, 6% is o.k. or should I go down? I once tried 4,5%, but I couldn't handle it, just ruptured a few times and I had a nice puzzle.
if you wait a little I will send you the exact protocol of Baus and Pfitzner TODAY!!