Luciferase Assay for Physarum Amoebae

Tim Burland (Last Update June 27, 1994)

(For more details, see Burland & Bailey, 1995, in the list of References)

1. Make a lysate of 5x10⁷  transformed amoebae

• Count luc-transformed cells (haemacytometer)  about 3-6 h after electroporation then spin down 5x10⁷ cells for 2 min at 200 x g
• Resuspend in 1mL BSS
• Spin down 25 sec. in microfuge at 7000rpm
• Aspirate supernatant with a Pasteur pipette
• Place cells on ice for 1 min
• Resuspend cold cells in 100mL of ice-cold  Luc Lysis Buffer by aspiration;  incubate on ice for 2 min
• Quick-freeze in dry ice / EtOH bath.
• Either assay immediately or store at -70^oC until ready to assay

2.  Assay lysate for luciferase

The key components of the assay reagent are Coenzyme A, Luciferin and ATP;  luciferase is detected by luminometry, which measures the photon emission that accompanies the oxidative decarboxylation of Luciferin.

• Bring required volume of  Luc Assay Reagent to room temperature (usually 100uL per assay) in the dark
• Meanwhile, prepare luminometer and check background of [Luc assay reagent + Luc lysis buffer] blank
• Measure 20uL cell lysate into a luminometer tube
• Add 100uL of Luc assay reagent and immediately place into luminometer to measure RLU (Relative Light Units).

At least 10⁵ RLU should be detected following electroporation with 1 ug pPardC-luc DNA (Burland & Bailey, 1995)

To save reagents, 10uL cell lysate can be assayed with 50uL Luc assay reagent.

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Last modified: Monday, December 2, 1996