Pull down assay---- to study which proteins interact with the "bait" - Which technique is good? (Dec/06/2006 )

Hi, I will carry on experiement to study what proteins will interact with my protein of intrest--Cofilin 1.

My intention is to pull down the protein complex use Cofilin as a bait, and run SDS-PAGE gel of the complex, then to cut bands out, and do MS-MS to identification the proteins.

I heard of several techniques:
1. GST pull down (Pierce Cat#21516)
2. HSI spin trap (GE)
3. HA-tag pull down
4. Immuno precipitation

Any one got experience of such experiment.
Please give me some suggestion.


Thank you

Hello!
I had tried immuno precipitation. For this experiments, first of all, you need good antibodies. For Cofilin.
There is a very easy kit for using, I think form Sigma, where you bind the antibody on particles that have proteinA, than this antibodies bind the protein of interest. the next step is that you run over your support your mixure of interest and pull of the complexes. This is one way.
the other is to bind your protein on magnetic paricles and than run your mixure of proteins over this particles . but for this you need a very clean and pure protein Cofilin (and possibly not denaturated).

In both cases there is the posiblity that not all of the proteins from the mixure (the one that are supposed to bind your protein) interact with Cofilin due to negative sterical interactions.
Also be careful with antibodies, be sure that bind not denaturated Cofilin!

Good luck with your experiments!!!!!!!

Thank you very much.

May I know the name of the product from singma?

What kind of antibody you consider good? mono-clone antibody?