purification of prot interacting with a probe in EMSA - (Jan/18/2006 )

I wonder if it's possible to elute the protein interacting with the DNA probe in gel shift assay in order to sequence it by ms/ms or malditof

pardon my ignorance, but could you not ID the protein if you used ChIP?

Isn't that basically a pull-down assay that involves the same principles as EMSA?

my understanding is pretty rough and I might be off base?

I think that the Chip is a technique making it possible to determine the DNA sequence with which a protein interacts. To do that you need a specific antibody directed against your protein wich you want to identify its DNA target.

Hi Fredo and Ami,
My old boss has a protocol for purifying proteins bound to an oligo. You basically use a biotin-labelled ds oligo and affinity preciptitate it using streptavidin-agarose beads. You can then Western blot the sample. I'm not sure if it would be pure enough for the mass spec. but maybe after western/2D gels perhaps.
The method is used in the following reference. Oncogene. 2005 Nov 21 Regulation of cyclin D2 and the cyclin D2 promoter by protein kinase A and CREB in lymphocytes. White PC, Shore AM, Clement M, McLaren J, Soeiro I, Lam EW, Brennan P

All the best,
Ceri

Thanks a lot Ceri, i have already try that but it doesn't work, but it was a home made protocol. I will also try with this protocol
have a nice day

I think a more complete version of the protocol is in Interleukin protocols, a methods book edited by Luke A J O'Neill and Andrew Bowie, if you can get hold of it. When you say it didn't work, did you mean you couldn't pull down the protein or you couldn't elute it off? I think in the protocol I mentioned the beads are boiled in gel sample buffer to elute the proteins because it's meant for use in Westerns. I'm sure you could probably substitute a high-salt wash as you would use in the nuclear extraction to elute the proteins and dialyse the sample to remove the salt.

Let me know if you want a more detailed protocol with your e-mail address. I could probably get hold of one.

All the best,
Ceri

some of those damn scientists are so clever...

thank you for the information, Ceri; I have teased that one around in my head before and it is nice to know there is a method. we do many gel shifts around here and after a couple of those you start to think it would be very useful to be able to ID the protein without a ss

hello Fredo,
I saw yr post today and was wondering if you have already tried and succeeded eluting the binding protein. I have started doing EMSA, got the shift also and thought about doing the same thing a while ago. Was delighted to see someone's similar views. Please share yr experience with me. Also I got the crisp bands in gel shift assay using 200 bp probe but could not get competition to work. It is always smeary and inspite of being specific to the probe, gives unusual high intensity shift instead of reversing it. Can you explain why?
Thanks,
uhsna.