in vitro translation problems

Will Fuller wf10000 at cus.cam.ac.uk
Mon Dec 23 05:51:55 EST 1996


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




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