Problems using pGEX
(by shaakanin from yahoo.com)
Sat Nov 1 17:53:35 EST 2008
1. Re: Problems using pGEX
Ok this could be long:
There are so many different problems you could be having with this expression.
Some of these are going to sound like stupid questions, however, I think this is where to start:
#1) You said you had problems with cloning, are you sure your gene is present?
#2) Have you also sequenced your gene to make sure you don't have nonsense mutations?
#3) Are you sure you're in the right reading frame? You say you "don't get" your protein, but does that mean you're getting GST and not your protein, or you're not getting GST either?
#4) What growth conditions have you tried? (Media, temperature, induction, additives) Just because the plasmid is there, and you have the right sequence and you're in the right reading frame- doesn't mean that your protein will automatically be there.
Make sure 1-3 are answered and let me know what growth conditions you've tried and let's go from there. I am happy to help with any protein problem- and I understand just how frustrating something like this can be. You may also have a toxic protein, or heterologous isseus, which may have to be solved a different way.
Sometimes yeast, insect cell, gram positive bacteria any of these can work while e coli does not.
Cheers and best wishes
Jess Grad. Res. Asst. University of Texas- Austin Institute for Cellular and Molecular Biology Department of Medicinal Chemistry -Hook 'em Horns!
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