Good day everyone,
I am having some issues with co-immunoprecipitations and I was wondering if maybe someone could help me resolve the issue.
Currently, I have cells which are expressing a FLAG tagged version of the SNARE Vamp3 (hence referred to as Flag-Vamp3) and the goal of what I'm trying to achieve is to immunoprecipitate Vamp3 and identify its interacting partners. Thus far, I am optimizing my protocol and I can successfully pull down Flag-Vamp3; however, I cannot co-immunoprecipitate any proteins with it. I know that Syntaxin 4 and Snap23 are well-known SNAREs that associate with vamp3 and that have been shown to co-precipitate in the past and yet I cannot see this with my co-IPs.
I have tried multiple lysis buffers and I found 2 that were working just fine and managed to Co-immunoprecipitate the upper mentioned proteins with Flag-vamp3 a couple of times consequently. But after a certain time, I wasn't able to reproduce those results with either of the buffers. I did not change anything in my protocol, I've tried to redo the lysis buffers as well as their various components freshly and I always end up pulling down Flag-vamp3 but not the prey proteins.
I would like to know what options do I have to potentially fix this situation or understand at the very least where the problem may lie.
Protocol for IPs is as follows (Briefly):
Obtain fresh lysates, pre-clean them by 1h incubation with Prot G coated agarose beads at 4 on rotating platform, transfer lysate to agarose beads pre-incubated with the anti-Flag antibody and incubate 2h30min at 4 on rotating platform, wash the beads 3 times with lysis buffer and store the samples at -80 until SDS-PAGE gel analysis (usually the next day). All manipulations with cell lysates are done on ice.
Thank you in advance
Dear,
Roman Telittchenko, I believe that the choice of beeds was flawed. Check the organism where the FLAG protein was raised, such as Mouse or Rabbit, if you want to use it to draw down your target protein. Because you are employing Sepharose-G beads, which are more particular to any antibody generated in mice, you won't receive any pull-down products if the IgG you were using was the Rabit specific FLAG ab. As a result, I advise using Sep-A beed before pulling down with FLAG (rabbit-specific). Please verify the pH of your IP-Lysis buffer as well; it should be between 7.2 and 7.5. This, in my opinion, will resolve your issue.
Best
SUBHAJIT
If your problem isn't with your pulldown coming down (are you getting at least 5-10% of your bait to come down?), lots of possibilities. One, can you reproducibly detect your prey in your cell lysates? If not, your prey's expression may fluctuate in time. If you could detect your preys previously and can't anymore, consider using a recombinant option (always useful to validate native interactions). Good luck!
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