Luciferase activity by scintillation counting?

BACKD at QUCDN.QueensU.CA BACKD at QUCDN.QueensU.CA
Fri Sep 4 08:20:15 EST 1992


Steve,

     I strongly recommend getting a luminometer to do luciferase
assays, particularly if you plan to assay many samples. You can get
a simple luminometer such as the LKB 1250 for < $5000. We used a
scintillation counter in our early assays. It was acceptable, but
a little awkward to use. Please consult the following reference:

Nguyen, V.T., Morange, M. & Bensaude, O. (1988) Firefly luciferase
luminescence assays using scintillation counters for quantitation
in transfected mammalian cells. Anal.Biochem. 171, 404-408

     In the traditional luciferase assay light is generated in a
pulse lasting several hundred milliseconds. Accurate measurements
required that injection be done in the luminometer and the area
under the pulse peak integrated for assay results. K.V. Wood
developed a method that converted the assay from a "flash" to a
"glow" assay (see below). The light production is very stable and
decays with a half time of 5 minutes. This allows measurement of
light intensity in a luminometer very similar to spectrophotometric
measurements. Alternatively, counts per unit time in a
scintillation counter will give similar results. This makes for a
very "gentlemanly" assay. We calculate it costs about $0.04 to $0.1
per assay.

Luciferase "Glow" assay:

5X Cell lysate buffer:

     -125mM Tris-PO4, pH 7.8
     - 10mM CDTA
     - 10mM DTT
     - 50% glycerol
     -  5% Triton X-100

Luciferase assay buffer:

     -20mM Tricine, pH 7.8
     - 1.07mM (MgCO3)4 Mg(OH)2 . 5 H2O
     - 2.67mM MgSO4
     - 0.1mM EDTA
     -33.3mM DTT
     -270Um Coenzyme A, lithium salt
     -470uM Luciferin
     -530uM ATP

Assay:

-Rinse cells 2X with PBS
-Add a minimal volume of 1X lysis buffer, ie 250ul per 60mm dish,
incubate 10 minutes at room temperature.
-scrape cells into a microcentrifuge tube, spin 10 sec in microfuge
-decant supernatant and use for assay. Luciferase is stable for
extended periods when stored at -70C.

-Mix 20ul of extract with 100ul of assay mixture. Mix then place in
luminometer or scintillation counter.

-Use 1X lysate buffer containing 1mg/ml BSA to dilute samples.


Don Back
Department of Biochemistry
Queen's University
Kingston, Ontario



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