Should blocking step be included after X-linking in Co-IP?

[jongleur]

Dear all,
I have a general question regarding the protocol of crosslinking antibody to beads for co-immunoprecipitation and eluting with pH3 glycine for mass Spec.

Since antibody is more expensive than protein A beads, I guess most people would incubate antibody with excessive amount of protein A beads. Will the unbound protein A co-ip non-specific immunoglobins from whole cell lysate, which can be eluted by glycine and detected as heavy chain and light chain in SDS-PAGE gel? Is there a way to block the unbound protein A after crosslinking antibody to beads?

Thanks.

[CrowSan]

Hi it has been a while since I did co-ip with protein A beads but if I remember the main source of protein contamination after elution was from the ab I cross-linked to the beads. To get around this , after cross-linking the ab I washed with low pH elution buffer to remove non-crosslinked ab (prior to adding to cell lysate). Then I re-washed the beads several times in PBS I think then added them to my lysate.
Even then I did see some of my ab chains in the final elution after IP (although I was looking at very long exposures).
I would certainly block the beads. I think most background is proteins non-specifically binding to areas of the beads that don't have ab attached rather than to protein A itself.

[jongleur]

Hi CrowSan,

Why do you 'think most background is proteins non-specifically binding to areas of the beads that don't have ab attached rather than to protein A itself ' ?
Is it because the amount of immunoglobins can be detected from cell lysate is considered low enough?

Is there a efficient way to block beads? If I use BSA to block the beads, should I add crosslink reagent after blocking? otherwise the BSA will be eluted by low pH glycine.

[mdfenko]

If I use BSA to block the beads, should I add crosslink reagent after blocking? otherwise the BSA will be eluted by low pH glycine.

bsa won't bind to protein a. protein a will only bind immunoglobulins.

[relaxin]

According to the manual (see link below) of Pierce® Crosslink Immunoprecipitation Kit, 10 ug of IgG are used for 20 ul of Protein A/G agarose beads. No blocking of excess Protein A/G was mentioned. You can block the Protein A/G with non-immune IgG, after removing your specific antibody, and then cross-link all IgG molecules to the beads. This may prevent subsequent binding of nonspecific IgG from you lysates and co-elution of this nonspecific IgG with your specific antigen. Alternatively, you can preclear your lysates with Protein A/G agarose beads, before incubating them with the specific antibody-agarose beads.

http://www.piercenet.com/instructions/2162134.pdf