Co-Immunoprecipitation

[bioquest]

Hello everyone,
I have a very basic question about co-IP. I have to co-IP interaction of this protein A and see what does it interact with. I am transfecting it in HEK 293 cell lines and trying to co-IP with Myc and HA (two tags attached to this protein A). My question is if I IP with Myc and western with HA can I see proteins interacting with this protein. Do I need serum as a negative control even if I use two different antibodies to do IP and western?

Thank you so much for your help.

[relaxin]

Since both Myc and HA tags are on the protein of interset, if you IP with anti-Myc and western with anti-HA, you will only show one band of the protein of interest, and not the interacting partner.

[CrowSan]

Normally to demonstrate that two proteins interact (such as Protein X and Y) a cell line (293 cells) is transfected with two constructs: Ha-tagged-Y and Myc-tagged-Y. Then one is pulled out (IP'd) with one tag. Probing a western blot with an antibody to the second tag indicates if interaction occurs or not. Checking for co-ip of native proteins with a "bait" protein is possible but is very hard to do as in a cell the amounts of protein that will interact in a co-ip is very low (as they are not over-expressed like the "bait" protein). You also need to "know" what interacting proteins to look for and have an antibody to match.

[relaxin]

If you are confused with CrowSan's post, it is a typo, it should be "Ha-tagged-X and Myc-tagged-Y".

If the interacting partner for your protein of interest is unknown, you can still identify it. Most people run the co-IP on SDS-PAGE, locate and excise the unknown band after silver staining, and do trypsin digest for mass spectrometry analysis. I cannot give you the details, as I do not work on this stuff. You can send your sample to the core facility of your university for custom service.

[bioquest]

Do you mean SDS-PAGE on co-IP by co-IP on SDS-PAGE? THat is do co-IP and run SDS-PAGE, do silver staining and cut out the band, digest and send for mass spec.

Thanks for your reply.

[relaxin]

What is the difference between "SDS-PAGE on co-IP" and "co-IP on SDS-PAGE"?

To me, both mean the same thing; do co-IP and run SDS-PAGE, do silver staining and cut out the band, digest and send for mass spec.

[CrowSan]

If you are confused with CrowSan's post, it is a typo, it should be "Ha-tagged-X and Myc-tagged-Y".

Yep. It was a typo