no expression in pET 21 vector - (Dec/19/2008 )

hii everyone,

hope someone can help me with my expression problem,

i meet problems with expressing my new psychrophilic eukaryote protein in pet 21 vector. until now no band was detected in both SDS PAGE and western blot. the vector was transformed in BL 21 (DE3) and origami 2 (DE3) host. I grow my cultres in 37, 30 and 15 deg for 2, 4,6,8 and 24h. Just nothing. please someone help me because i'm worried if i change the expression system to pichia there is still no result for the expression.


help me,

mazila

I know it sounds obvious but is your IPTG fresh? We had a project student in the lab who was seeing no expression for the first 4 weeks of her project and then finally we discovered see was using a stock of IPTG that had been in the freezer for a few months. As soon as she made a fresh stock she got expression straight away!! Doh!

How do you induce the culture and how do you prepare samples for SDS-PAGE and Western? There could be many reasons for no expression, too little information
www.yoproteins.com