Hey everyone,
I am performing immunoprecipitation of an antibody of interest from brain and spinal cord tissues. The antibody in monoclonal and is not coupled to the beads we are using. The protein eluent is later on analyzed using SDS-PAGE and sent for mass spectrometric analysis of interacting partners of my bait protein. Unfortunately, in the last two attempts, our mass spec collaborators spotted many antibody fragments in the IP samples, which were not appearing (at least not to a huge extent) in our bead control. The suspicion is that the fragments are coming from the monoclonal antibody used for the IP and that it was somehow truncated during the processing of the samples (for example during elution using loading buffer and boiling).
Does anyone have experience with how to avoid such problem or find an alternative way to elute the proteins from the beads?
Thank you very much in advance!
I agree with Sebastian.
You'll almost always get some antibody fragments in your samples, expecially when the antibody is not crosslinked to your beads and especially when you're using denaturing conditions for elution.
Furthermore, if you want to use the same beads more than once, you could try to elute your proteins from the antibody with a low pH buffer instead of a denaturing loading buffer.
As an elution buffer I used "0.1 M sodium citrate; pH = 2 - 3". Antibody fragments were almost absent afterwards, compared to samples where the antibodies were not crosslinked to the beads.
I worked with "Dynabeads Protein G" to which I crosslinked my IgG antibody (not monoclonal in my case).
Boiling is certainly going to release antibody by damaging the resin, breaking disulfide bonds between the heavy and light chains as well as breaking of Asp-Pro bonds due to heating in SDS sample buffer. If you insist on boiling your sample consider using a lower temperature loading buffer like the Novex LDS buffer and keep the heating to a minimum (no more than 5 minutes). Your best bet for a clean elution would be to use a peptide of your epitope (most IP kits include these) which could compete off your precipitated epitope. Peptide would run at the electrophoretic front of your SDS-PAGE and should be out of the way of your MS analysis. A pH shock (as mentioned above) would also be a more gentle option- glycine is a popular reagent for this. Keep in mind that some of these chemical elutions can cause your SDS-PAGE to run crooked if you load too much so you may need to clean them up with a buffer exchange or a precipitation step (but cross that bridge when you come to it).
First of all, thank you very much for your helpful answers!!
Sebastian Schmitt
: The antibody components are a tremendous problem for us since they mask less abundant interactors (it could be that in this specific interactome trial the IP was not so efficient to begin with, but the antibodies are making the analysis even harder).Would you recommend to us the other elution options in combination with in-solution digestion rather than in-gel digestion for mass spec?
And one last thing, for the people who couple the antibodies to beads themselves: do you have some recommended protocol to share?
It would be very appreciated!
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