Streptactin Column Regeneration

Streptactin is a mutated streptavidin protein so treat it like you would any other protein!''' The most stringent way to wash is to use 0.5M NaOH. Fill the''' column and allow to drain by gravity. You must immediately neutralize the beads with PBS or other neutral pH buffer after the NaOH has drained. Do 2 column volumes to be sure beads are in the correct pH. Sometimes Rick includes 1% Triton X-100 with the 0.5M NaOH to help remove any hydrophobic gunk.

Optionally, clean with HABA buffer: 50mM Tris, pH8.0, 150mM K-ace, 1mM EGTA, 1mM HABA. HABA is orange dye in solution but turns bright red when it binds to the streptactin. It binds in the biotin/strep-tag binding pocket, so column will only turn red if the streptactin protein is still competent to bind strep-tag. Wash with 1 column volume of HABA buffer and then need to wash column with multiple columns of PBS or other buffer to remove the HABA dye, until beads are mostly white again. Sometimes they remain a little pink and that is ok.