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IP - crosslinking antibody to dynabeads vs. NHS-acitvated beads?

IP MS

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#1 Euterpa12

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Posted 29 August 2013 - 07:04 AM

Hi guys!

 

I'm trying to identify protein-protein interactions by doing an IP for my target protein and then identifiying interacting partners w/ MS. For optimization I've been doing WB to prove that I precipitate the target protein and silver stainings to see if additional proteins are pulled down. 

 

For the IPs, I used Dynabeads Protein G (Invitrogen), but I get a huge signal from the antibody heavy and light chain in both the silver staining and WB. I'm guessing this would also be a problem w/ the MS so I would like to try coupling the antibody to the beads.

 

My question is whether I should try crosslinking the antibody to the protein G dynabeads, or would it be better to use NHS-acitvated magnetic beads that will directly covalently bind the antibody? 

 

One of the concerns about the NHS-activated beads is that they would bind the antibody wherever there are primary amines, which could result in the lower affinity or avaliability of the Fab region for the antigen. Whereas with crosslinking to protein G, the antibody is already correctly positioned to interact w/ the antigen, which would result in a better precipitation (better meaning more protein)

 

Does anyone have any experience/thoughts/recommendations on the type of beads I should use?

 

Thanks a lot!

j.



#2 Missle

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Posted 30 August 2013 - 04:01 AM

If I'm understanding correctly, the problem is that the antibody is being 'let go' from the Protein G during your process.  Can you alter your process to prevent this?  If your antibody is a mouse or human it should bind to the protein G very tightly at neutral pH but it will let go with a significant drop in pH.  If I knew what your conditions I may be able to help with what is causing your antibody to be let go..... 

 

What you said about a NHS linkage is true - it will cause some antibody molecules to be unable to bind your target or not be able to bind it as tightly but potentially this could be overcome by building a bigger column?



#3 miST32

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Posted 23 February 2014 - 01:02 AM

Hi Euterpa,

I've been working on IPs with both approaches.  Generally, a very good IP antibody doesn't seem to perform better or worse with cross-linking vs. direct NHS beads.  Poor antibodies seem to work better with the cross-linking approach though.  So does antisera - you may need to purify antisera via dialysis or immunoaffinity if you want to use NHS beads.


In my experience so far, the cross-linking still leaves a bit of IgG in the eluted fraction.  I don't notice as much with the NHS beads.







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