Luciferase/CAT assay cell extracts?
cocea at bisance.citi2.fr
Fri Jul 28 10:35:11 EST 1995
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".
I have two questions:
1. Has anyone already done this, I mean prepare both cell extracts with the
Promega Reporter Lysis Buffer, then do a Luciferase assay on some of the
extract and a CAT assay on the rest of the extract? Does it work? Have you
compared the readout of the CAT-assay done this way with the read-out you
have for a CAT-assay done by using a freeze-thaw prepared cell extract?
2. Has anyone performed both assays with a cell extract prepared with the
freeze/thaw method? Promega says freezing/thawing greatly reduces the
luciferase activity after two cycles...As a matter of fact, someone in my
lab did it with sevaral vectors; the problem is the activity seems to be
reduced for some vectors but not for all of them. What do you think about it?
Thank you very much for your help.
cocea at citi2.fr
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