Protein purification after in vitro transcription/translation using Roche RTS100

Kyle Legate legatek at mcmail.cis.mcmaster.ca
Tue Nov 26 17:46:49 EST 2002


On 26 Nov 2002 aennsche at yahoo.com wrote:

> Hello!
> did anyone use the protein in vitro transcription / translation system RTS100 by Roche. i would
> like to express my kinase (which is not possible in bacteria, yeast and plant cells) in order to do
> kinase assays and pulldowns. i was wondering if the yield is high enough to be able to purify the
> desired protein and then use it.
>
This sounds like an incredibly expensive approach. In my experience the
amount of protein made in an in vitro retic system is in the neighbourhood
of 1-5 fmole/microL using an SP6 promoter. You might blow the budget
obtaining enough protein to do kinase assays.
	If your protein does not depend on eukaryotic modifications for
activity, you might think about using an E.coli S30 lysate system which
can be made in house. Check out
http://www.science.mcmaster.ca/biochem/faculty/andrews/lab/projects/methodsandprograms/labman/ec1.html
for the protocol. You will still require quite a large reaction volume to
get enough protein for useful experiments, but at least it's relatively
cheap.

... . . .  .  .  .    .    .    .     .     .     .      .      .      .
legatek at mcmaster.ca		Kyle Legate            legatek at hotmail.com

   Tower of Tongues:Thursday PM:10:30-11:30 EDT:http://cfmu.mcmaster.ca
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