Purification of a functionally active protein kinase from bacteria

Frank Fuerst fant.1 at gmx.net
Tue Apr 25 05:25:50 EST 2000


Andrew J Ippolito schrieb:
> 
> 
> according to the postdoc, this works.  Not for me.  And it is growing VERY
> frustrating!  Can anyone help?  I thank the list in advance for any input.

BTW this is not a list, it's a usenet newsgroup...

What do you mean with "this works...not for me"?

Have you done SDS-Page with all the fractions? Can you see induction
of your protein anywhere?
Possibly you have a problem with the bacteria, or impure DNA (e.g.
plasmids religated without the insert coding for your protein). I
would:
- make miniprep DNA of the respective colony, digest and look if the
plasmid is correct (it has been sequenced anyway, has it?)
- test for the best growth temperature (compare induced and
identically treated non-induced cells, lysed in some quick way and
immediately applied to a SDS-gel). You might need to lower the
temperature if your protein is destabilized.
If you can see your protein beeing induced, you should monitor where
you "loose" it, so you'll find the point where you have to change the
protocol, or be more careful with it.

Hope this helps, Frank
-- 
Die Verwendung von mehreren Ausrufezeichen macht die Aussage nicht
ausrufender sondern ausufernder. [Michael Bauer in dnq]




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