peptide expression problem - (Jul/23/2006 )

Hi,
I've been trying to express a peptide of 20 amino acids, as a fusion to a His tag. Expression is pretty low and I guess i should expect that, I've looked up numerous papers and posts and quite a lot of people have this problem.
but now out of the blue, it just stops expressing for a while -at least it doesnt show iup on my westerns. then it appears again after i have tried everything and i'm about to commit suicide. . Right now i'm in one of those disappearing stages. has anyone else had this problem?
i really cant depend on its whims to continue work. this sort of thing looks like it could go on forever.
I'd really appreciate any light on this. Thanks

-soraya-

Do you always use the same gel percentage? And the same transfer condition? Can you think about something you changed?

-dnafactory-

QUOTE (soraya @ Jul 24 2006, 03:23 AM)

. Right now i'm in one of those disappearing stages. has anyone else had this problem?
i really cant depend on its whims to continue work. this sort of thing looks like it could go on forever.

I'd really appreciate any light on this. Thanks

Hi.
I had a problem like that a few years ago. It seems that even though my construct was expressing (we tested this by in vitro transcription/translation), it was so small that the cells thought it was a misfolded peptide, and prompty digested it!
You might have more joy if you make a fusion to something else, such as GST. This is what I did for my thesis work with a 45mer peptide, and I got beautiful expression and purification.

-swanny-

hi ,
yes i kept every thing exactly the same. and when that didnt work i tried changing conditions too. i think i'll also try what swanny suggested. thanks so much.

-soraya-

我使用的pET32载体, 效果很好

-ccemd-