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-   -   Problems with Immunoprecipitation and Cross-linking (http://www.molecularstation.com/forum/immunoprecipitation-forum/3623-problems-immunoprecipitation-cross-linking.html)

kiki06 07-23-2008 11:58 AM

Problems with Immunoprecipitation and Cross-linking
 
Hi,
I am conducting IP on a 70kDa factor. I am trying to do IP and pull down with
Dynabeads -Protein G from Invitrogen. It is not working at all. Does anyone have any experiences with pulls downs with IP then western blot / detection?

How can i be sure the crosslinking worked?

thanks

danfive 07-23-2008 07:38 PM

Re: Problems with Immunoprecipitation and Cross-linking
 
Are you crosslinking your Ab to the bead? If so you can test that by quenching the reaction (usually Tris buffer), washing, eluting (perform usual elution step), equilibrating then doing a Protein Quant assay. If you have protein means your Ab eluted and crosslinking wasn't 100% effective. If no protein is detected by Protein Quant Assay your Ab is linked to the beads.

kevin_a 07-23-2008 08:10 PM

Re: Problems with Immunoprecipitation and Cross-linking
 
Kiki,

Macroboy had similar problems with the experiment not working.
To test the crosslinking he just ran the elution fractions on gel and did the Western looking for heavy chain. (not many people are willing to go through that much trouble)

However, since your protein is 70K, you could probably get away without doing the crosslinking. Especially if you have a monoclonal antibody. Sometimes they do not take to kindly to being crosslinked.

K

marcroboy 08-12-2008 02:05 AM

Re: Problems with Immunoprecipitation and Cross-linking
 
Hi Kiki,
I guess if I checked this forum more often I could've replied earlier, I'm not sure if this is still troubling you.
Anyways, like kevin_a said, I was trying to do a similar pull-down experiment with dynabeads protein G. My cross linking reaction did work except that I couldn't get method of eluting just the protein. I tried Citrate ph 3 buffer in their manual which gave me no protein, however when I boiled the same beads in SDS loading buffer I saw the protein on the western blot gel.


However, I ended up forsaking the cross-linking because like Kevin said,if you can resolve the protein from the antibody heavy chain, you shouldn't need cross-linking. And in my case, I had a negative control antibody(preinjection serum) which I ran along that clearly told me that the additional band is the protein that I pulled down.

marcroboy 08-12-2008 02:28 AM

Re: Problems with Immunoprecipitation and Cross-linking
 
Oh also just in case anybody else is watching this thread, does anyone reuse these dynabeads, I tried to reuse the beads I did IP with earlier(they were boiled in loading buffer) and the resut didn't come very good. The manual says they could be reused for up to 5 times but I guess it depends on the method of elution? These beads are pretty expensive so I don't really feel like wasting them because for my next procedure I plan to do a lot of IPs.

And another question I have regarding the storing of Protein G-Ig complex. For all of the IP I ran, I used fresh immunoprecipitated products but I am wondering if protein G-Ig complex can be stored in say the washing buffer in -4 for later use(to capture my protein), would Ig degrade or dissociate over time? I wanted perform Casting on it which requires multiple cycles of IP hence if I can make a whole bunch of protein G-Ig complex in one batch, it would save me a lot of time in the long run.

samriddha 09-04-2008 04:37 PM

Q: Problems with C0-Immunoprecipitation
 
hi All,
Expt: to check interaction between protein A and B, i'm pulling down Protein A and blotting for Protein B. my one sample has both my proteins A and B (with GFP and Myc tags) and i am using protein B only as the negative control.
But in the western, i can detect signal even in my negative control, which is, only the protein B!
my guess is the protein B is sticking to the Magnetic beads (NEB,protein G beads) that i am using.

TO get over the non-specific binding issue, i have tried salt washes,with 300mM,400mM and even 600 mM Nacl post-IP but
that seems to disrupt my actual binding while the band in the negative control still persists.

will any one of you have any idea/s how to go about this issue??
ALso, does Dyna1 magnetic beads work better than the NEB protein G magnetic beads??
hope to get some feedback.
thanks a lot in advance,Sam.

kiki06 09-04-2008 09:40 PM

Re: Problems with Immunoprecipitation and Cross-linking
 
thanks everyone for the input.

Yes I heard you can reuse the beads but i would only reuse them for the same protein sample. I will try to find a protocol for reusing Dynabeads and other magnetic beads. :notworthy:

marcroboy 09-26-2008 02:24 AM

Re: Problems with Immunoprecipitation and Cross-linking
 
Quote:

Originally Posted by kiki06 (Post 9164)
thanks everyone for the input.

Yes I heard you can reuse the beads but i would only reuse them for the same protein sample. I will try to find a protocol for reusing Dynabeads and other magnetic beads. :notworthy:

hi, so have you found it? I tried to reuse the beads, I didn't do anything special with it, I just REUSED it lol. It looks like it's been working.

Roald 12-12-2008 10:31 AM

Re: Problems with Immunoprecipitation and Cross-linking
 
Hi all

Dynabeads Protein G can be reused up to 5 times, but only if you use non-denaturing elution buffer, e.g. 50 mM Glycine, pH 2.5-3.0. If you elute with denaturing conditions, e.g. boiling in LSB, you will also denature the protein G on the beads... I never reuse beads for a different protein sample, as there is always a risk of remnant Ab and or ligand on the beads.

We store Ig-coupled Dynabeads Protein G at 4 degrees in PBS, pH 7 with 0.1% Tween 20 to prevent aggregation. Not all Ab's can tolerate storage at 4 degrees though.

Crosslinking the antibody to the beads often decreases binding efficiency, sometimes totally. We only crosslink when the size of the target can not be resolved from the Ig heavy chain on WB.

In our experience, many antibodies do not work for IP, even if they work well for other aplications. If at first you don't succeed.... try a different Ab to your target... :-)
Roald

jay1iisc 07-06-2009 04:30 AM

Re: Problems with Immunoprecipitation and Cross-linking
 
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