Toxic protein for E. coli (M15)

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Fri Sep 6 08:36:26 EST 2002


Thanks for sharing the info Frank.  Very informative.  I'll just add
a few observations of mine

>
>To check _total_ expression, we routinely test whole bacterial extract:
>1ml of induced culture, spun down, resuspended in 100ul of SDS gel
>sample buffer, sonicated to reduce viscosity (important!), and 15ul per
>lane analysed by SDS PAGE.

If you don't have a sonicator available, you can pass the sample up
and down about 5 times through a 22 ga needle attached to a 1 ml
syringe.  A bit messy but quite effective in reducing the viscosity.

>
>Changing temperature down to 28° is also often recommended. For us, this
>did not change anything. To get results, the temperature must be lowered
>to below 18° to really change the bacterial metabolism in a way that
>_may_ affect expression levels and solubility of a protein. But still,
>this is guarantee that it works. In the two cases where I tried it, the
>protein simply was not expressed any more...

I do know of at least one case where shifting the temp from 37 to 28
made a big difference in the amount of enzymatically ACTIVE protein
being expressed. It's easy enough to try, and knowing that it worked
at least once is enough to make me want to try it!
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