35s labled gst fusion protein

Dr Engelbert Buxbaum engelbert_buxbaum at hotmail.com
Wed Mar 5 03:59:33 EST 2003


fisher wrote:

> If I want to get this kind of protein in E.coli, how to do it?
> Does it need in vitro translation?


Buy some Translabel (a mixture of 35S methionine and cysteine) and a
sulfur-free medium (for mammalian cell culture these are readily
available, I'm not sure for E. coli). Grow your bugs in an o/n culture,
spin them down and wash with sulfur-free medium. Resuspend them in that
medium and add Translabel (working in a fume cupboard, just to be safe).
Incubate for about an hour, spin (remember that the medium is hot). Now
you can wash with buffer and lyse the bacteria, or you can wash them in
normal medium and give them a chasing periode, depending on the purpose
of your experiment.

There is one alternative though. Radioactive methionine decomposes
relatively rapid, forming reactive byproducts (aldehydes?) that can
label purified proteins (Bowder et al., . Anal. Biochem 204 (1992)
85-89, Kalinich & McClain: Anal. Biochem. 205 (1992) 208-212). That way
you avoid large volumes of radioactive waste.



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