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Problems with biotynylated protein binding to monomeric avidin - (Oct/07/2013 )

We are trying to purify a 20kD soluble protein following its expression in BL21-BirA. While a control protein (Avitag-GFP) is successfuly purified on monomeric avidin column, our protein always goes through the column and does not bind. Did anyone encounter such a problem?


Hi Stive.  I haven't encountered this exact problem, but I have attempted other affinity chromatography columns that didn't work.  Are you able to detect the 20kD-biotin protein in the lysate and the flow through in a manner that would indicate is actually is biotinylated?  Is your control protein expressed in the same BL21-BirA cells?