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Co-IP: Prey Protein Present in Neg and Pos Eluates

platelets proteins IP Co-IP

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5 replies to this topic

#1 vh04x

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Posted 26 August 2012 - 12:47 PM

Hello everyone!

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

ProA:
In - +
x - x (these x's represent bands on a western, dashes represent a lack of one Posted Image)

However, the Co-IP seems to pull out ProB in the negative eluate, and I can't figure out why

ProB:
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

Edited by vh04x, 26 August 2012 - 12:51 PM.


#2 ascacioc

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Posted 28 August 2012 - 01:27 PM

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.

Andreea

#3 vh04x

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Posted 28 August 2012 - 06:42 PM

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)

#4 ascacioc

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Posted 28 August 2012 - 10:50 PM

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 :)

Andreea

#5 vh04x

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Posted 29 August 2012 - 07:40 AM

Thanks :)

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

#6 ascacioc

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Posted 29 August 2012 - 11:28 AM

If it is the kit, it is more stable that what I thought initially. No clue, sorry :(





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