Hi all, I am performing an IP using 20AA biotinylated peptides as bait and recovering peptide-protein complexes using streptavidin beads. I elute the proteins using heat, SDS and mercaptoethanol, however, my peptide still stays in a complex with protein and this is interfering with downstream experiments. Any suggestions on getting rid of the peptide, and not the big proteins?
Thanks in advance!
It depends on your downstream applications, but sometimes I would use a Glycine-HCl pH = 2 solution to get rid of every interaction ( i used it for antibody-antigene interactions), normally it would do the work. However, depending on the affinity of your protein for your ligand, you can increase salt concentration in your buffer or dialyse ( not sure that dialysing would work though)
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