Hey guys,
do you know another eluent for a streptavidine column than 8 M guanidine-HCl? I need to catch some biotinylated proteins but I'm not sure whether pH 1.5 will also disrupt my biotin from the protein (I still need the biotinylation). Denaturation of the protein is not a problem.
Does anybody know? Thanks =)
No, but I though about. Did anyone use a high biotin concentration and was successful?
If you really think the 1.5 will disrupt the bond you can still use 6 M guanidine or 8 M urea and just keep the pH neutral. The guanidine should still remove the protein from the streptavidine beads and not affect the bond to biotin. If you need it acidic you can control it above pH 3 and should be fine.
Even though it appears that you do not require your protein to remain active, I would rather go for a reversible avidin-biotin interaction as available with monomeric avidin. Here, free biotin is used for elution. Due to the strong interaction, other avidin-biotin com flexes cannot be broken with avidin or biotin as eluants.
Try the link:
https://www.thermofisher.com/br/en/home/brands/product-brand/dynal/streptavidin-coupled-dynabeads/elution-of-the-streptavidin.html
Based on the recommendation by Elio Baba, I would say using 10 mM EDTA along with 6M guanidine pH 8.0 would work fine. You can always remove the guanidine downstream using either filtration cutoff like centricons or bind it to a C18 spin column and then elute with a volatile organic if you want to do MS etc.
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