EMSA Supershift for STAT3 - (Aug/16/2006 )

Hi Biomaus,
I add the ab (1-2µg) so in the Sc-482x format (200µg/0.1ml) that will be 0.5-1µl to the binding reaction containing nuclear extract before I add the probe. I incubate for 20mins at room temperature then incubate with the probe for 20mins at room temperature. My previous lab incubated for 30mins for both but 20mins seems to be fine. I use my standard binding buffer (5X binding buffer 50mM HEPES pH7.9, 250mM KCl, 50% Glycerol, 1mg/ml acetylated BSA, 5mM DTT, 1mM PMSF so 1X final concs. 10mM HEPES pH7.9, 50mM KCl, 10% Glycerol, 0.2mg/ml acetylated BSA, 1mM DTT, 0.2mM PMSF plus 1µl poly dI:dC (1mg/ml) per binding reaction)and I think a 6% gel should be fine.

There's an example in this paper using a 8% gel (6% probably would have been better).
Fielding CA, McLoughlin RM, Colmont CS, Kovaleva M, Harris DA, Rose-John S, Topley N, Jones SA. R
Viral IL-6 blocks neutrophil infiltration during acute inflammation.
J Immunol. 2005 Sep 15;175(6):4024-9.

Maybe the 4% gels are best for making jigsaws.

All the best,
Ceri

Thanks Ceri, I will try it this way. I just phoned with Sc and convinced them to send me the ab for free, so I hope, it will work then and with your nice help!

No problem. One more thing before I forget. If you publish using a Santa Cruz ab if you list the catalogue number in the material and methods there was a offer of a free ab. Don't know if they're still doing that.

Ceri

Pfitzner's Protocol. Since you are from Tübingen I assume, that you understand german. If not I will translate if necessary.

Hi Jou,

thank you for the protocol. I'm german, so no problem with the language I will try this next week when I have got the new antibody.

I'm with Ceri

also, I had a hell of a time getting a supershift once and was going crazy...added some BSA to the binding buffer and it finally worked.

so, perhaps switching to Ceri's recommended binding buffer?