Protein unavailable for IMAC binding
(by ben.long from yourfinger.anu.edu.au)
Thu Feb 28 17:34:19 EST 2008
I'm expressing several proteins in BL21(DE3) using 0.5 mM IPTG for
induction. One protein is proving a little difficult in that it is
successfully extracted into the soluble fraction but only a relatively
small proportion of this is subsequently bound to IMAC. I say relatively
small in that a huge amount does not bind to IMAC but recovery of pure
protein is still quite good. It would seem that a large amount of the
protein is forming soluble, yet unbindable (occluded tag), aggregates. I
wondered if using lower IPTG concentrations would reduce the total
protein concentration and therefore the likelihood of aggregation or is
there another approach? I do not wish to use denaturing agents (e.g.
urea or guanidine) during purification as the protein is bound for
crystallisation. Any ideas?
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