Description

The SuperLight^TM Luciferase Reporter Gene Assay is based on the quantitation of luciferase expression in mammalian, yeast or E. coil cells, using luciferin and ATP as substrates. The reaction results in light production which can be conveniently measured on a luminometer.

   Reaction scheme:

                                     Luciferase

ATP + D-luciferin + O₂                     oxyluciferin + AMP + PP_i + CO₂ + light

This bioluminescent reporter gene assay is extremely sensitive and is especially suitable for quantifying luciferase expression in recombinant cells. This ultra-sensitive, homogeneous cell-based assay only requires adding a single reagent to the cells and measuring the light intensity after a short incubation step (2 minutes). Assays can be performed in tubes, cuvettes or multi-well plates. All kit components are compatible with culture media and with all liquid handling systems. With an extended luminescence emission kinetics (half-life 40 min), the SuperLight^TM luciferase assays are especially suitable for high-throughput screening in 96-well, 384-well and 1536-well plates. In addition, the reagent provided in the kits has been formulated for maximum sensitivity, reproducibility and long shelf-life. Applications for this kit include gene regulation studies and high-throughput screening of gene modulators.

Key features

High sensitivity and wide detection range: detection of as little of 2 fg luciferase and as few as 4 cells. Plus, the emitted light is linear over seven orders of magnitude.

Compatible with routine laboratory and HTS formats: assays can be performed in tubes or microplates, on LJL Analyst, Berthold Luminometer, Top-Count, MicroBeta counters, chemiluminescent image plate readers (CLIPR/LeadSeeker). Assay reagents compatible with all liquid handling systems.

Fast and convenient: homogeneous “mix-and-measure” assay allows detection of luciferase levels within 10 minutes. The optimally combined reagent system allows a single addition step, and simultaneous cell lysis and detection.

Robust and amenable to HTS: Z’ factors of 0.6 to 0.8 are observed in 96-well and 384-well plates. Can be readily automated on HTS liquid handling systems.

Applications

Gene Regulation: gene expression level, characterization of promoter activity, modulation of gene expression by receptors, transcription factors and small molecules.

Drug Discovery: high-throughput screen for gene modulators.

Kit Contents

Storage conditions. Store the Reagent in the provided amber tube at -20°C and the Assay Buffer at 2-8°C.  Shelf life: 12 months after receipt.

This protocol can be downloaded online at www.bioassaysys.com.

Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.

ProcedureS

The   SuperLight^TM  Luciferase   Reporter  Gene  Assay  is  based  on  the bioluminescence generated during the luciferin/luciferase reaction. The reconstituted reagent has been optimized to combine cell lysis and detection into one single step. Phenol red in culture media does not interfere in this assay. All data in the Technical Notes were obtained in media containing phenol red.

Important: fresh reconstitution of the Reagent in Assay Buffer is recommended, although the reconstituted Reagent may be stable for up to 4 weeks when stored at -20 °C.

Procedure using 96-well plate:

1. Plate and culture cells (80 mL) in white opaque 96-well tissue culture plates. Typical culture medium contains DMEM, 10% fetal bovine serum and antibiotics (penicillin/ streptomycin, gentamycin, etc). Amino acids and other nutrients can be added to the culture medium. Assays can be performed on either adherent cells or cells in suspension. The cells can be either stably or transiently transfected with the luciferase gene. Culture volume can vary from 50 to 100 mL, although 80 mL is used in this protocol. Blank control wells containing no cells should also be prepared.

2. Add test compounds and controls to