NovaBright™ Chemiluminescent Reporter Gene Assays

Illuminate the Path to Biodiscovery

Reporter gene assays are invaluable for studying the regulation of gene expression by cis- or trans-acting factors, or for measuring activation of cell signaling pathways. In these assays, the reporter gene acts as a surrogate for the coding region of the gene under study. The reporter gene construct contains one or more gene regulatory elements being analyzed, the structural sequence of the reporter gene, and the sequences required for the formation of functional mRNA. Following introduction of the reporter construct into cells and experimental treatment, expression levels of the reporter gene are monitored by quantitating the reporter protein enzymatic activity, which is used to characterize regulatory sequences, transcription factor activity, cell signaling, or signaling pathways.

NovaBright™ Chemiluminescent Reporter Gene Assays

Assays based on enzyme-triggered 1,2-dioxetane chemiluminescent substrates offer several important advantages compared to alternative systems. The inherent low background and high signal intensity result in exceptionally high assay sensitivity, enabling femtogram levels of enzyme detection with a wide dynamic range of typically up to 5–6 orders of magnitude. Rapid signal generation together with extended-glow light emission kinetics permit fast assay completion, often in less than 1 hour, with flexibility in read time and compatibility with batch-mode processing. These reporter gene assays, discussed below, are widely cited in scientific literature for their numerous applications in research and high-throughput screening applications. See Table 1 for a comparison of NovaBright™ chemiluminescent reporter gene assays offered by Life Technologies.

Table 1. NovaBright™ chemiluminescent reporter gene assays.

* Secreted placental alkaline phosphatase.

Phospha-Light™ and Phospha-Light™-EXP SEAP Assays

Secreted placental alkaline phosphatase (SEAP) is secreted by cells directly into the culture medium, enabling the nondestructive assay of culture medium samples, preserving cells for additional assays and enabling time-course monitoring of gene expression. NovaBright™ chemiluminescent SEAP reporter gene assays exhibit remarkable sensitivity and ease of use.

The Phospha-Light™ and Phospha-Light™-EXP SEAP reporter gene assays are used in transiently transfected cell lines and primary cells, stably expressing cell lines, and serum from transgenic animals for gene expression assays, gene knockdown/RNA interference readouts, virus expression assays, and protein secretion pathway analysis. Both systems offer highly sensitive assays that can be completed typically in less than 1 hour and preserve valuable cells for further experimentation.

The next-generation Phospha-Light™-EXP SEAP reporter gene assay has a higher signal intensity and higher signal-to-noise performance than the original Phospha-Light™ assay system (Figure 1). All reagents are provided ready to use. The assay can be performed directly on the culture plate or on supernatant transferred to a separate plate. The Phospha-Light™-EXP assay is ideal for enabling multiparameter detection from a single culture plate, demonstrated by quantitation of SEAP expression from cell culture medium aliquots, followed by measurement of cell viability in the cell culture plate with the PrestoBlue™ Cell Viability Reagent (Figure 2). Time-course monitoring of gene expression is possible by removing multiple culture medium samples from a single cell culture plate (Figure 3). Since this leaves the cells intact, they can undergo multiple experimental treatments, be processed for imaging, or be subcultured post-analysis for SEAP expression.

Figure 2. Multiparameter assay capability with the Phospha-Light™-EXP SEAP assay. NIH/3T3 cells were transfected with pCRE-SEAP (cAMP-inducible SEAP expression) using Lipofectamine® LTX reagent with PLUS™ reagent. Cells were treated with forskolin for 24 hr to induce cAMP production. Samples of cell culture media were removed and SEAP induction was measured with the Phospha-Light™-EXP SEAP assay. Cells were then assayed for viability using the PrestoBlue™ cell viability and fluorescence detection.

Figure 3. Time-course expression analysis with the Phospha-Light™-EXP SEAP assay. NIH/3T3 cells were transfected using Lipofectamine® LTX reagent with or without PLUS™ reagent with pCMV-SEAP (constitutive SEAP expression) with high or low final DNA concentrations. Culture media samples were serially removed from wells at the indicated times after transfection and assayed with the Phospha-Light™-EXP assay. Data were obtained from a single 96-well plate. and expressed as signal (RLU) from wells containing transfected cells divided by signal from wells containing mock-transfected cells.

The Gal-Screen® and Galacto-Star™ Reporter Gene Assay Systems

The Gal-Screen® and Galacto-Star™ assays set the standard for ultrasensitive quantitation of β-galactosidase (β-Gal) in both mammalian and yeast cells. Chemiluminescent assays exhibit up to 1,000-fold higher sensitivity than colorimetric assays, enabling picogram to femtogram levels of detection. Unlike other luminescent β-Gal assays, our assays provide direct detection of β-Gal activity and do not rely on other enzyme cascades. Both assays can be used with β-Gal reporter as a readout for traditional gene expression assays in many cell types and tissues, or as a functional readout for viral function, immune cell activation, cell death, and mRNA processing. In addition, they are used for highly sensitive detection for β-galactosidase enzyme complementation assays used for monitoring of protein–protein interactions, protein translocation, and receptor dimerization/activity, including high-throughput screening of compounds for receptor activation. High sensitivity makes these systems ideal for detecting low levels of expression and for transfection normalization.

The Galacto-Star ™ assay provides a simple procedure: following culture media removal and cell lysis, reaction buffer containing chemiluminescent substrate is added. The Gal-Screen® assay provides the simplest protocol, designed for use in high-throughput screening workflows: a single reaction buffer that provides cell lysis and chemiluminescent substrate is added directly to cells in the presence of culture medium (Figure 4).

The Luc-Screen® Reporter Gene Assay System

The Luc-Screen® assay conveniently couples cell lysis in the presence of culture medium with a high-sensitivity assay that exhibits extended-glow light emission kinetics. The assay system is designed for mammalian cells with maximum assay flexibility in a high-throughput format. It has been used for gene expression assays, compound screening, and large-scale promoter function assays. Light signal can be measured in as little as 10 minutes following reagent addition and remains nearly constant for up to 4–5 hours, providing flexibility in the time between reagent addition and measurement. The high sensitivity of the Luc-Screen® assay system enables detection of as little as 50 fg of luciferase, with linear signal over 6 orders of magnitude (data not shown).

The Dual-Light® Reporter Gene Assay System

The Dual-Light® assay system is designed for rapid and sensitive detection of both firefly luciferase and β-galactosidase reporter enzymes in a single sample. This microplate assay system is widely used with transfected cell lines and primary cells. The Dual-Light® assay system is designed for transfection normalization, using two reporters: one as an experimental reporter (typically luciferase), and the other as a constitutively expressed transfection control (typically β-galactosidase) to accurately quantitate activity from experimental reporter constructs.

Luciferase activity is detected via an enhanced “flash” luciferase reaction; then, following a 30–60 minute incubation and addition of a light emission accelerator, β-galactosidase activity is measured. The combined assay, completed typically in 1 hour, detects femtogram levels of each enzyme, enabling detection of low levels of reporter produced by weak promoters or poorly transfected cells. With over 6 orders of magnitude, the wide assay dynamic range offers measurement over a broad range of reporter enzyme expression levels (Figure 5).

NovaBright™ Reporter Gene Assays.*

For Research Use Only.  Not intended for any animal or human therapeutic or diagnostic use.