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co-Immunoprecipitation


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#1 Priya914

Priya914

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Posted 17 September 2010 - 11:50 AM

Hi all,
I badly need some help here. I started working on co-IP experiment since end of May with no luck of clear answer so far
I am trying to detect if Prot A binds to Prot B. Both of them are transcription factors and our lab has shown that both of them bind to a very small DNA sequence which made us assume that they interact.
Now I pulled down Prot A and can successfully do it. The antibody used for pulldown is Mouse monoclonal and I use mouse IgG as control (can I use rabbit IgG or any other IgG instead of mouse ?) For detecting Prot A and Prot B after IP I use a rabbit polyclonal. I can see Prot A pulldown clearly. However, in my blot for Prot B, I see a band at the same mol weight as Prot B.
The same thing occurs when I pulldown Prot B with rabbit polyclonal and blot for Prot A with mouse monoclonal. In this case I used Rabbit IgG as control IgG. I see a band at exactly same molecular weight i control lane.
IgG light and heavy chains are not a problem since these proteins are 75 and 82 kDa.
The thing that confuses me the most is I am able to see these proteins interacting when I do GST pulldown experiment but no luck with co-IP.
Do you absolutely have to have co-IP result to show an interaction ? Is GST protein pulldown not enough ? Should I do mass spec on the band ? Can I use a beads only control instead of IgG ?
Your help is very much appreciated. Thanks for your time.




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