CAT assay problem!
Manuel Maria SIMON
Simon at mail.boku.ac.at
Fri Mar 17 11:26:37 EST 1995
jr at dna.bio.warwick.ac.uk wrote:
> Dear All,
..The problem I have is that after heating the cell extract
> for 15 min at 65C, the extract "goes cloudy" and the ppt can be spun down. My
> question is what is this ppt. and will it interefere in the subsequent CAT
> assay and can I prevent it coming through. ...
If you´ r doing the assay with TLC separation you might get a smear!
Therefore I switched to an alternative protocol.
I´m using 3H Acteyl-Coenzyme A (e.g. Amersham TRK.688 9,25 MBq/ml) and
cold chloramphenicol. Proteinextracts can be made also by two freeze
thaw cycles or also by hypotonic swelling. The you extract your reaction
with Dimethylether. You count the organic phase.
This is much easier than any protocol I know.
Contact me if you want to kow details.
Manuel M. SIMON PhD
Center of Applied Genetics
Univ. of BOKU Vienna
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