Assuming all other conditions are equal, any idea what the relative efficiency of immunoprecipitating a protein using noncovalently bound antibody vs covalently crosslinked antibody is?
Yes it will almost certainly alter the efficiency of immunoprecipitation (in a negative way), but nobody can predict relative efficiencies. This is trial and error, if you have enough pure antigen you will be able to determine relative efficiencies yourself with dilution series. I prefer to have my antibodies catching their antigens on their own...and then bringing them down with protein A sepharose using low speed centrifgation (100g) or Protein A magnet beeds (even lower speed). Why do you want to cross-link them?
Crosslinking through lysine residues, the most common method, randomly orients the antibody attachment point, rendering some of the antibody unable to bind to the antigen. Capturing the antibody with Protein A, which binds to the Fc region, avoids this problem and makes sure that all the antibody is oriented in such a way that it can bind to antigen.
Thanks guys, this was helpful. I'm using protein A/G magnetic beads in order to immune precipitate a protein of interest to screen for interacting partners by mass spec. I don't want excessively abundant peptides from my antibodies to swamp the signal from less abundant proteins. I'm using protein A/G beads because I'm using three distinct antibodies that recognize three regions of my protein and I want to reduce variability due to differences between nonspecific binding to protein A vs G. I'm performing coIP-MS from knockout mice as well as a negative control.
Adam, might incubating the antibody with the beads overnight prior to crosslinking increase the proportion of antibodies in the correct orientation?
If you are asking about using chemical crosslinking to covalently attach the antibodies to protein A/G on beads, then the answer is probably yes. If you are asking about chemical crosslinking antibodies to beads without protein A/G, then the answer is probably no.
Thanks, yes. I'm using protein A/G dynabeads. The rationale being I'll allow the antibodies to obtain the proper orientation noncovalently overnight and perform the chremical crosslinking the following day.
I would guess that the concentration of crosslinker and duration of the reaction could be important parameters. Too much crosslinker reaction with the antibody could reduce its affinity for antigen.
Hi,
an alternative to cross-linking is to directly couple the prim Ab covalently to the Dynabeads M-280 Tosylactivated or Dynabeads M-270 Epoxy. Also the Antibody Coupling kit and co-IP kit can be used which includes buffers for coupling and for optimizing the conditions for co-IP (co-IP kit). this ensures functional Abs on the bead surface (see; Albert et al. 2007 (Nature 450, 683-694)
kind regards
ketil.
I have also similar experiment design and faced with similar problem (don't want to elute antibody with 8M urea before sending for LC MS/MS analysis). I have tried 1hour incubation of Ab (V5) with dynabeads protein G and used BS3 1.75mM for 30min, which certainly affected my Ab-ag interaction (I couldn't see the same band pattern on western as I saw without cross-linking). I have now ordered Pierce NHS activated magnetic beads and waiting for those to try. A close chemistry friend said this shouldn't affect the Fab region.
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