The Turner BioSystems' 20/20ⁿ Luminometer in combination with Promega's Steady-Glo® Assay kit provides a convenient, rapid, and sensitive procedure for quantifying gene expression. Transcriptional regulation, coupled to the expression of a luciferase reporter gene, is regularly used to study a wide range of biological events in cultured cells. Luciferase is an ideal reporter because of the absence of endogenous luciferase activity in mammalian cells, and the functional enzyme is created immediately upon translation^1,2.

The Steady-Glo^® Luciferase Assay System has been developed specifically to maximize the sensitivity of the assay reagent while providing a luminescent signal half-life of approximately 5 hours. The light signal can be measured between 5 minutes and several hours after adding assay reagents. The Steady-Glo^® Reagent is widely used in the pharmaceutical and biotechnology industries. It is compatible with commonly used culture media for mammalian cells (RPMI 1640, MEMa, DMEM and Ham's F12) and tolerates phenol red and organic solvents.

The superior sensitivity of the 20/20ⁿ Luminometer combined with the effectiveness of the Steady-Glo^® Reagent permits detection of very low levels of luciferase activity. The 20/20ⁿ Luminometer can detect as little as 1X10^-19 moles luciferase enzyme. (Note: Assay limited sensitivity.) Measurements are linear from 1X10^-19 to 1X10^-11 moles luciferase or 8 orders of magnitude (Figure 1). All tests were conducted using Steady-Glo^® Luciferase Assay Kit (Promega Catalog# E2520) and purified recombinant firefly luciferase enzyme (Promega Catalog# E1701).

Figure 1. Steady-Glo^® Assay was performed on the 20/20ⁿ Luminometer using Promega's Steady-Glo^® Reagent kit and recombinant luciferase.

2. MATERIALS REQUIRED

• 20/20ⁿ Luminometer
  (P/N 2030-000)
• 1.5 mL microfuge tubes
• Steady-Glo^® Luciferase Assay kit (Promega Catalog#'s E2510, E2520, E2550)
• p200 pipette and pipette tips

3. EXPERIMENT PROTOCOL

3.1 Reagent Preparation

Steady-Glo^® Substrate: Use as supplied. Store at -20°C, where it is stable for up to 6 months. The substrate may also be stored at 4°C for up to 1 month.

Steady-Glo^®Buffer: Use as supplied. Store below 25°C.

Steady-Glo^® Reagent: Transfer the contents of one bottle of Steady-Glo^® Buffer to one bottle of Steady-Glo^® Substrate. Mix by inversion until the substrate is thoroughly dissolved. Use reconstituted reagent on the same day it is prepared or store at -20°C for up to 2 weeks.

Note: The temperature of the Steady-Glo^® Reagent should be held constant at room temperature while quantifying luminescence, since luciferase activity is temperature dependent. Reagent stored frozen after reconstitution must be thawed below 25°C to ensure reagent performance. Mix well after thawing. The simplest method for thawing is placing the reagent in a water bath at room temperature.

3.2 Instrument Setup

3.2.1 Turn ON the 20/20ⁿ. A 5 minute warm up period is recommended, but not necessary.

3.2.2 Touch "Run Promega Protocol" from the "Protocols" menu.

3.2.3 Select "SteadyGlo" from the list of Promega protocols. The Parameters screen appears next with pre-programmed settings that are optimized for the Steady Glo Assay.

3.2.4 Touch "Advanced Options" to access "Replicates" and "Automatic Blank Subtraction" features (Optional).

3.2.5 Touch "OK" to go to the "Home" screen.

3.3 Sample Analysis

3.3.1 Remove the cell cultures from the incubator.

Note: For maximum reproducibility, equilibrate cell cultures to room temperature before adding reagent.

3.3.2 Add a volume of the Steady-Glo® Reagent equal to that of the culture medium.

3.3.3 Wait a minimum of 5 minutes to allow for sufficient cell lysis, then transfer the sample to a 1.5 mL microfuge tube for analysis.

3.3.4 Insert the tube into the 20/20n using the microfuge tube holder and touch "Measure Luminescence" to begin measurement.

4. REFERENCES

1. Ow, D.W. et al. (1986) Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science 234, 856-9.

2. De Wet, J.R. et al. (1987) Firefly luciferase gene: structure and expression in mammalian cells, Mol. Cell. Biol. 7, 725-37.

5. ABOUT PROMEGA CORPORATION

Steady-Glo is a trademark of Promega Corporation and is registered with the U.S. Patent and Trademark Office. Orders for Promega's products may be placed by:

Phone: (800) 356-9526
Fax: (800) 356-1970
E-mail: custserv@promega.com
Internet: www.promega.com
Mailing Address:
Promega Corporation
2800 Woods Hollow Rd.
Madison, WI 53711
USA

6. ABOUT TURNER BIOSYSTEMS

Orders for Turner BioSystems' products may be placed by:

Phone: (408) 636-2400
Toll Free: (888) 636-2401 or
Fax: (408) 737-7919

Contact us via our contact form
Internet: www.turnerbiosystems.com

Mailing Address:
Turner BioSystems, Inc.
645 N. Mary Avenue
Sunnyvale, CA 94085

CAUTION: The lyophilized Steady-Glo^® Substrate contains dithiothreitol (DTT) and is therefore classified as hazardous. The reconstituted reagent is not known to present any hazards as the concentration of DTT is less than 1%. However, we recommend the use of gloves, lab coats and eye protection when working with these or any chemical reagents. Promega and Turner BioSystems assume no liability for damage resulting from handling or contact with these products.

[Back to Top]