pET expression system

Marc Visconti scontz at passport.ca
Thu Feb 23 22:43:44 EST 1995


I'd like to thank everyone for being so kind as to send me their 2 cents
worth regarding my problem.  However, I realize I may not have fully
explained my problem.  The term "increadible induction" was in reference to
my Western blots.  With the other expression systems used, I never saw a
clear cut difference between my induced and non-induced fractions.  I made
a conclusion that my protein is soluble by running soluble and insoluble
lysates after sonication.  Virtually all of my product, when detected with
the S-protein Western kit, was soluble.   
 
Currently my protocol requires plating transformed cells on kanamycin
plates (50 ug/ml), picking a colony, growing o/n to an OD-600 of 1.5- 2.0. 
This is used as innoculate into fresh media (LB for now) with 50 ug/ml
kanamycin.  These are grown to OD-600 0.4-0.6, and then induced with 1mM
IPTG for 2-3 hours.  These are then spun down, and resuspend in Sample
running buffer with SDS, boiled and run on SDS-PAGE.  Am I doing something
wrong or missing something?  Thank you in advance for all of your help. 
 
--Marc Visconti 
scontz at passport.ca 
marcvis at resunix.ri.sickkids.on.ca



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