in vitro translation problems

Mircea Podar podar at utsw.swmed.edu
Tue Dec 31 12:56:43 EST 1996


In article <59lo8b$j82 at lyra.csx.cam.ac.uk>, wf10000 at cus.cam.ac.uk (Will
Fuller) wrote:

> Hi,
> 
> I'm trying to do a bit of basic biochemistry and I'm having untold
problems getting it to work. I'm using the Promega all in one in vitro
transcription and translation system. I can see the protein expressed fine
but I can't get any post-transational processing when I add my canine
pancreatic microsomes.
> 
> The plasmid I'm using expresses in mammalian cell systems, so the
necessary signal sequences are there. I'm wondering if there is some
species specificity problem (human protein, canine microsomes)? 
> 
> If the protein is processed correctly I would expect to see a 20kDa
change in molecular weight. I've tried all the controls provided with the
microsomes without success, and I've tried a large range of amounts of
microsomes to add (o.3ul to 2.5ul). Can you help?? If so, please e-mail me
at wf10000 at cus.cam.ac.uk
> 
> Thanks for your hel



Just a thought. Do you know for sure the nature of your protein
post-translational modification? There are many other modifications besides
phosphorylation or glycosylation and the microsomes might not be able to do
everything. I never used the microsomes, they might be just fine for
"ordinary" modifications (did you have a positive control?), but the
protein you're dealing with could be special.
Good luck,
Mircea

podar at utsw.swmed.edu



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