Imran Mungrue
imran.mungrue@utoronto.ca
14) Luciferase Assay
*Aspirate media from cells.
*Rinse cells in PBS 3 times.
*Add 250ul Harvest Buffer and scrape off cells
Harvest Buffer:
(Store @ -20 or 4degC)
500ul Tris/MES (1M, pH7.8)
50 Triton X-100 (20%)
10 DTT (1M)
9.44ml water
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10ml
*OPTIONAL Freeze pellets for up to 3 weeks @ -80degC, but do not vortex to keep cells intact.
*Transfer cells to eppendorf tube, vortex for 10sec to lyse cells.
*Spin down at full speed in a micrfuge 10min @ 4degC.
*Remove 200ul supernatant, add 15ul Luciferase Cocktail
Luciferase Cocktail
(Store at -20degC)
900ul Tris/MES pH7.8 (1M)
180 MgOAc (1M)
96 ATP (500mM)
24 water
------
1000
*Read samples in luminometer with Luciferin Injected
180ul KPO4 (1M, pH7.5)
36ml water
5mg Luciferin
Tris/MES
(pH to 7.8)
12.1g Tris Base
21.3g MES
100ml water