protein-protein interaction-plz help - (Jun/06/2007 )

I am trying to detect interaction between two proteins by Immunoprecipitation but till now not suceessful. For these two proteins I have found interaction by Two-hybrid analysis. I am confirming this result by IP technique.

I can detect protein band in the lysate ( supernatant) but can not detect in beads. I am using protein A beads for this purpose.
Can anybody suggest how can I improve my techniques?

Is there any other convenient method to prove this binding?

plz help

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What buffer are you using? Protein:protein interactions can occasionally be very sensitive to their buffer.

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swanny is right, if your buffer's salt concentration is high, then you reduce the number of protein that your protein interacts. because, ionic strenght distrupts the prot-prot interaction. good luck
one way to overcome this problem can be to decrease the salt concentration of your buffer . by the way, I use the same bead. if you read the description, I you'll get what I mean.

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Thx Iayda and Swanny for ur comments.

First I used 1% Digitonin Buffer.

Composition of this buffer is given below : First I make 2x BN-PAGE buffer as follows:
100mM Bis-Tris ,1.5M e-amino-N-caproic acid , pH 7.0

Then I make 1% Digitonin Buffer solution with 1x BN-PAGE buffer.

To make cell lysate this 1% Digitonin buffer was supplemented with proteinase inhibitor.


Since I was not successful , I changed the buffer.

Here is the composition of Buffer I used later :

50 mM Tris-HCl(pH 7.5)---------

100mM NaCl ----------

2mM MgCl2 ----------

0.5% Sodium deoxycholate--------

1% Triton X100 ---------

10% Glycerol

This buffer was supplemented with proteinase inhibitor while making cell lysate. I tried it once. But yet not get the precipitation of the interacting partner in western blot analysis.

I am very very frustrated. Do u have any more suggestions?

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i would suggest you to try the BC buffers.
I showed interaction with BC150buffer or even BC300 buffer

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thx fred

would u tell me more about the composition of this buffer u suggested?

thx in advance.

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I've spent the last two years doing co-IPs and my boss describes them as one of the most difficult biochemical assays so don't give up! First, your buffer has a touch more detergent than mine. If you are getting no signal from the beads you need to use a less stringent buffer... get the interaction first, then get it clean. I use a simple NP-40 lysis buffer: 150mM NaCl, 50mM Tris (pH 8.0) and only 1% NP-40 but when I was first looking for the interaction it was only 0.5%. How do you make your lysates? I never vortex or sonicate my samples or vortex the bead washes. Are you sure protein A beads are best for binding your antibody (ie: protein A barely binds mouse IgG1 whereas protein G is much better)? Are you sure the IP is working? Do you have something you know binds your protein that you can check for? Do you have a cDNA you can express in the cells before the IP? Some interactions can only be detected by western blot when one or both of the proteins are overexpressed.

Another way to check for this interaction is to do an in-vitro pull-down. It's easier to detect an interaction in this assay but is often criticized for being an artificial environment. Plus you may have to make reagents (ie: GST-tagged protein) in order to go this route. However, an interaction I'm working on which barely shows up in Co-IPs is very obvious in a GST-pulldown with radiolabelled TNT protein or cell lysates.

I spent my first year of graduate work chasing potential interactions from a 2-hybrid. Once I showed my boss that the interactions didn't even show up in GST-pulldowns we moved on. You may have to face the idea that you are working on what is, in fact, a false positive hit from the 2-hybrid. 2-hybrids are messy and can result in many false positives... in fact one of my titles for a lab presentation was "Friends don't let friends do 2-hybrids". I lost a full year of work and a post-doc in my lab lost two years chasing interactions from 2-hybrids which looked so promising.

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i prepare BC0 buffer and add KCl from a 3M stock solution.
BC0 composition is :
20 mMHepes, pH7.9 / 0.2 mMEDTA / 20% (vol/vol) glycerol/0.5 mMphenylmethylsulfonyl fluoride/3 mM dithiothreitol

wang and roeder also used it for purification of recombinant

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thx fred for ur information.
regards

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thx rkay447 for ur informative mail.

i am using rabbit antibody for IP so i use protein A beads .
for making cell lysate, previously I did sonication(20 sec on/off, 3 times).
But last time i vortexed the cells with lysis buffer + proteinase inhibitor for few seconds and then keep on ice for 10 min.

previously i tried to precipitate endogenous protein but i failed. Now i am trying to overexpress one or both protein ( which r GFP or HA tagged) and then immunoprecipitate.

I think IP is working as while trying to precipitate GFP tagged protein with GFP Antibody, I could detect GFP-tagged protein band in beads but I could not detect its associated HA-tagged protein band in beads.

I also dont vortex the beads while washing. I do gentle tapping.

I have few questions to u.

1. in ur NP-40 buffer do u use proteinase inhibitor?
2. u dont vortex or sonicate cells, then i am qurious how u make cell lysate?

thx in advance.

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