Co-immunoprecipitation - antigen elution without the antibody - (Apr/18/2007 )
I am trying to modify my protocol to elute my protein complex without the antibody. I am using sepharose beads with protein A, and with the small amount of antibody I have, cross-linking is not an option. I am using the indirect method (first - linking antigen to antibody, then - linking the whole thing to the beads). I don't want to denature the sample, because I don't want the antibody contaminating it. I have heard that high salt buffer might free my protein complex into the buffer, leaving the antibody on the beads. I know it depends on the specific antibody and the complex, though. Does anyone have any tips how to set the conditions of the elution? I would like to obtain my complex without the antibody in the process.
what antibody are we talking about here?
we currently use FLAG antibodies that you can elute with FLAG peptide for example
A more general method is elution by pH disruption, with 100 mM Glycine pH 3.0. The eluted protein should be immediately neutralized with 1mM Tris-HCl, pH 8.0
that works fine and MOST of the time you do not get the Ab
If you use an anti-FLAG antibody to capture your FLAG-antigen, you can elute with an excess of FLAG peptide,
However I donot completely agree about glycine pH3.0 elution, as it is how I elute my antibody from protein A sepharose when I'm purifying antibodies.
It's FLAGless and I know I can't use glycine. Is high salt of any use, 500 mM for example?
I would say that the affinity of the antibody for antigen is higher than the one of streptavidin for the antibody. So it's easier to elute antigen-antibody complex from streptavidin than to elute antigen from antibody.
Try to covalently bond the antibody, or try different system of detection, so you don't detect the eluted antibody (use a biotinylated primary antibody for western-blot, or use protein A-HRP instead of secondary antibody-HRP)