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pGEX fusions - GST band appearing.

gc genecutl at mendel.berkeley.edu
Tue Dec 19 16:27:42 EST 1995


In article <1995Dec18.162454 at molbiol.ox.ac.uk>, rpgrant at molbiol.ox.ac.uk
(Mad Dan Eccles) wrote:

#Members of the lab have been getting band(s) around 28 kDa on their gels when 
#doing GST-fusion protein preps.  The occurence of these bands varies,
sometimes 
#they're there, sometimes not.  There is a reaction at 28 kDa with anti-GST, so 
#it looks like the GST fusion partner. 
#
#I never had it occur until today, and it's not a single band, but a number of 
#distinct ones, all very close together on a 10% PA gel.  This is the first 
#time I've prepared this particular fusion.
#  We're all in PMSF up to the eyeballs, 
#keeping everything cold, there seems to be no common factor in the preparation 
#that correlates with the appearance of the GST band.  We sterile filter the 
#lysate before showing it to the glutathione sepharose, and generally use new 
#g-seph ( I did today).
#
#My feeling is that it is premature termination of translation, but I have not 
#yet done a Western to tell me whether this is so.  
#
#Does this ring any bells with anyone?
#
#TIA,
#Richard
#


   Yes, I see this a lot too.  It's definitely the GST half because it
   binds to glutathione beads.  And PMSF doesn't help because, at least in
   my case, it's already present in the E. coli before sonication.   One
   thing which does make a difference is the strain.  BL21 worked a lot
   better than DH5a.  I would think that, since this is occuring inside
   the cells, the less time the protein is in the cells the better.  So
   maybe higher IPTG concentrations for shorter times would decrease the
   amount of breakdown--although if there is a solubility problem with
   the fusion that may be exacerbated.

--gc



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