Hi :)
In addition to excellent replies already posted, a few thoughts:
1. Do the whole-protein MS of your stuff. Assuming that you get a spectrum then
a) If you are right and it is indeed your recombinant GST - you
will see a mass pattern that's interpretable via analysis of probable
cleavage
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- Reply message -
From: "Sebastiano Pasqualato"
Date: Fri, Mar 16, 2012 10:12 am
Subject: [ccp4bb] GST-fusion protein production in insect cells
To:
Hello,
Probably a stupid comment on my part, but anyways, make sure your strain has a
T7 polymerase! (I have forgotten before).
And I agree with the last idea that sometimes it is worth to just start from
scratch. New construct new vector. It may simply just work.
HTH,
Yuri
Hi Gerry,
In one case, I had a mutation in the ribosome binding site sequence. If you
haven't already checked, it might be worth checking whether the 5'- and 3'-
junctions close adjacent to the ORF are all correct to make sure the
transcriptional and translational elements do not somehow contain m
Hi,
if you use pLysS E coli strains and you're working with soluble cytoplasmic
proteins, a couple of cycles of freeze-thawing in LN2 will break your cells
pretty well with high protein recovery, without the need of extra exotic
chemicals added to your mixture. Just put some DNAse to avoid excess
v
Hi,
we also stain gels in microwave: Comassie with 5% acetic acid to stain
and just water for destaining. We keep the microwave under the fan
to avoid breathing acetic acid.
For E. coli cell disruption we are quite happy with Emulsiflex C3 by
Avestin.
In many cases proteins that undergo proteolys
