Protein unavailable for IMAC binding

Bean Long via methods%40net.bio.net (by ben.long from yourfinger.anu.edu.au)
Thu Feb 28 17:34:19 EST 2008


Hi all,

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?

Cheers,

Ben


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