pGEX fusions - GST band appearing.

Mad Dan Eccles rpgrant at molbiol.ox.ac.uk
Mon Dec 18 11:24:54 EST 1995


I think we've done this.... but...

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

-- 
Richard P. Grant MA DPhil      rpgrant at molbiol.ox.ac.uk
Nuffield Department of Obstetrics and Gynaecology, University of Oxford.
http://sable.ox.ac.uk/~lady0266
"Winds light to variable"



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