Luciferase/CAT assay cell extracts?

Bernard Murray murrayb at dc37a.nci.nih.gov
Fri Jul 28 12:50:00 EST 1995


In article <3vb03f$qnl at bisance.citi2.fr>, cocea at bisance.citi2.fr says...
>
>Hi-
>
>I intend to measure the efficiency of transfection in my CAT assays
>by cotransfecting a luciferase encoding plasmid (pCMV-Luc) with the
>plasmids that are the subject of my research.
>        However, I cannot use the CAT-assay cell-extract preparation 
>protocol with the luciferase assay. The Promega Tech Bulletin and a message
>I got yesterday from techserv at promega.com indicate that one has either to
>prepare two different cell extracts (i.e., use different protocols for the
>cell extracts in the two assays) or lyse the cells with the Promega Reporter
>Lysis Buffer which "allows luciferase, beta-galactosidase and CAT reporter
>enzyme assays to be performed from extracts of cells cotransfected with 
>vectors carrying these genes".

>Laurentiu COCEA
>cocea at citi2.fr
>

Yes, this "Lysis Buffer" had me wondering for a while.  I believe that
freeze-thaw or sonication are compatible with all three reporters but
the overall yields can be inconsistent.  I had a look around to try
and find detergents that are compatible with the reporter enzymes
(and that might be present in the Lysis Buffer).  My best guess would
be Nonidet NP-40 but I never got around to testing this.  I've never
possessed any of this mysterious buffer so I can't do it by analysis.
At the moment I do it using Triton X-100 and accept a slight reduction
in CAT sensitivity.
		Bernard

bernard at elsie.nci.nih.gov




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