IP - losing proteins from protein G beads - (May/02/2005 )
I seem to be losing protein from my protein G beads.
The short story is that we are using an assay which allows us to biotinylate sulfhydryl groups on proteins. The way we do that is to IP the epitope tagged protein - then perform a series of steps with which we block non specific sites (figuratively), expose the sulfhydryl groups, then expose to a biotinylated sulfhydryl specific reagent.
Briefly, after the IP, we expose the protein/beads to N-ethylmaleimide, which will bind to primary amines and sulfhydryls. then we wash the beads and expose to hydroxylamine, which frees certain sulfhydryls. then we expose to biotin-BMCC which is a sulfhydryl specific reagent. this process works quite well (or has in the past).
First, is anything known about hydroxylamine and its ability to interfere with antibody-antigen binding? I ask this because it looks like we lose protein following the hydroxylamine step.
Second - can I centrifuge the protein G beads too hard? I have used 14k RPM when I pellet the beads to wash them ... then i noticed that most protocols call for 2-5 k RPM ... I was wondering if that could also be a problem.
any advice would be great - thanks to all!
I have found that protein G beads are heavy enough to ppt without centrifugation, however, I also have used 14K centrifugation as well as 1K and this will not affect the protein G integrity, unless there is bacterial conatmination and they are being degraded (which is very rare). Looking at your appraoch in coupling your Biotinylation with IP it seems quite labroiuos (too many steps) and this might be the reason of lossing your protein, but before jumping to this conclsuion, I wonder if you have tested for the effeciancy of your IP, i.e. did you test for the Ip of your tagged protein before and after biotinylation to be positive that you had an IP problem. Good Luck!