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Should blocking step be included after cross-linking antibody to beads in immuno

crosslinking co-IP blocking protein A

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#1 jongleur

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Posted 20 June 2012 - 03:37 PM

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.

#2 bob1

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Posted 20 June 2012 - 06:01 PM

Yes, protein A will bind non-specifically to any antibody present. You can block the unbound protein A using IgG or serum that is not specific for your protein of interest. However, you need to use appropriate controls to ensure that any binding you then get is due to an interaction between your protein and its antibody.





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