proteolysis

Todd McGee scoop at leland.stanford.edu
Sat Dec 9 13:59:38 EST 1995


Before getting too involved in looking at proteolysis, you might take a
step back and look at what you're transcribing. SOme in vitro
translation systems are very prone to (multiple, spurious) internal
initiations. This is especially true if you don't have much sequence
upstream of the translation-start site. An easy way to see if this
COULD be the problem is to look at how many internal AuGs there are in
your transcript and determine if the too-small bands you're seeing
correlate with the size predicted from an internal initiation. 

If this doesn't look like it's the case and you've got plenty of
upstream sequence, then I'd consider PMSF (or APMSF since it's more
water soluble) and leupeptin to be great starting points. I believe I
also saw an ad for a protease inhibitor cocktail from Boehringer (I
think) that might be worth looking into if you don't want to fuss with
buying all the inhbitors and mixing them yourself. Hope this helps -

Todd McGee
Dept of Biol. Sciences
Stanford University
scoop at leland.stanford.edu


In article <klennon.179.30C83D6E at acs.bu.edu>
klennon at acs.bu.edu writes:

> I am currently performing in vitro transcription/translation studies in my lab 
> of an ER membrane-bound protein and am finding that the protein bands I see on 
> my SDS-PAGE are significantly smaller than I anticipated. I am in the process 
> of making sure my transcription reaction goes to completion, but am also 
> wondering if my protein is either running aberrantly or being cleaved.Does 
> anyone do in vitro translation in the presence of protease inhibitors? If so, 
> which one(s)? I've got PMSF, aprotinin, leupeptin, pepstatin at my disposal!
> 
> Thank you for any insight you could give me!
> Kelley



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