Co-IP: Prey Protein Present in Neg and Pos Eluates - (Aug/26/2012 )
New member; and in the few hours I've had to explore this forum, I have to say I'm very impressed with the wealth of knowledge that's available here. This is a wonderful resource.
Anyways, to the point:
I, like many other struggling grad students, am having difficulty optimizing a co-ip.
I'm trying to confirm a specific interaction between Protein A (ProA, bait) and Protein B (ProB, prey) in washed platelets. ProB was identified by LC MS/MS of excised bands from an affinity ligand pull down using ProA. IF shows that ProA and ProB co-localize relatively strongly. The only thing that remain is getting a solid Co-IP to confirm the interaction.
Lysis Buffers I've Tried:
25mM Tris, 150mM NaCl, 1mM EDTA, 1% NP-40, 5% Glycerol
25mM Tris, 150mM NaCl, 1mM EDTA, 1% CHAPS, 5% Glycerol
Tyrode's Buffer + 2mM CaCl2 + 2mM MgCl2 + 10mM Glucose + 0.5% Triton X-100
The IP works consistently for each of these buffers
Input (In): Platelet lysate in lysis buffer
- Eluate: Mouse IgG
+ Eluate: Mouse monoclonal anti-ProA
In - +
x - x (these x's represent bands on a western, dashes represent a lack of one )
However, the Co-IP seems to pull out ProB in the negative eluate, and I can't figure out why
In - +
x x x
Initially, I figured it was idiosyncratic for ProB. But I've repeated the above experiment with the listed buffers to test Protein C and Protein D (both of which are known binding partners cited in many papers). Same results. I can't for the life of me figure why. It couldn't be possible that ProB, C, and D all bind non-specifically to mouse IgG?
Thanks in advance for the advice guys
You don't have a ZZ domain in your prey proteins, do you? I mean you do not express your proteins from a plasmid that tags them with a ZZ domain? You do it with the native protein expressed from the chromosomes of your cells? I mean, the only thing that binds to IgG is a ZZ domain-tag.
Thanks for getting back to me.
No, none of ProA, B, C, or D have ZZ domains.
The Co-IP is performed from the lysates of platelets extracted from healthy donor blood. It's as physiological as it gets, I think.
Unfortunately, no one has managed to successfully make recombinant ProA, as it just falls apart when its synthesized out of the body.
ProA also has a nasty habit of binding to Protein A and Protein G, so this is Co-IP is performed with directly immobilized antibody (if that helps at all)
How do you immobilize the antibody on the beads? (short description pls; I want to check smth because I have a hint...I might be very off) I am also bumping your topic to be on top of the questions because you are without an answer for a few days
The beads have aldehyde groups on them that react with primary amines to form Schiff bases. NaCNBH3 is then added to reduce the bond to a secondary amine. Any spare aldehyde groups are quenched with reacting with Tris.
It's all part of the Pierce Co-IP (Direct) Kit
If it is the kit, it is more stable that what I thought initially. No clue, sorry