NovaBright Chemiluminescent Secreted Placental Alkaline Phosphatase (SEAP) Reporter Gene Assays

NovaBright™ Chemiluminescent Reporter Gene Assays

NovaBright Secreted Placental Alkaline Phosphatase (SEAP) kits contain the high-performance CSPD alkaline phosphatase substrate, Emerald luminescence enhancer, and a unique buffer system that specifically inhibits endogenous nonplacental alkaline phosphatase activity. The persistent glow of the assay signal permits the use of simple luminometers without injectors.

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NEW! NovaBright Phospha-Light EXP

SEAP Phospha-Light EXP and Original SEAP Phospha-Light Kit Benefits:

Designed to detect SEAP reporter gene expression
Measurement of either sample culture media or the total culture (cell and media)
No cell lysis steps—your samples remain intact
Easy and fast assay: <30 minute assay time, <1.5 hours to results
Wide linear dynamic range provides 3 orders of magnitude greater sensitivity than colorimetric methods
Proprietary buffer ensures low background—no signal from endogenous alkaline phosphatases
Persistent glow signal lasts for easy plate reading—no injectors required, assay is easily scaled up for higher-throughput plate formats
Compatible with Phenol red

New SEAP Phospha-Light EXP Assay Format Includes:

Ready-to-use CSPD substrate and next-generation Emerald enhancer
Only two assay reagents for simplicity
No dilution step
Significantly shorter heat-inactivation step

Performance

The new NovaBright Phospha-Light EXP kit provides higher signal intensity (Figure 1), higher signal/noise ratio, and low-end sensitivity compared to the original NovaBright Phospha-Light kit and other commercially available luminescent SEAP assay kits.

Figure 1. Higher signal intensity		Figure 2. Provides higher signal/noise ratio and low-end sensitivity.
Figure 3. The SEAP reporter gene allows for time-course and/or assay development studies that are inconvenient using assays that depend on cell destruction. Here, dose response of NFkB signaling in response to treatment with TNFa was monitored at different time points. HEK293 cells were transfected with a pNFkB-SEAP construct in a 24-well tissue-culture dish. At 48 hrs post transfection, media was removed and replaced with TNFa-containing media. Culture media was removed and assayed for SEAP enzyme activity using the NovaBright Phospha-Light–EXP Assay at the indicated time points.		Figure 4. Because SEAP reporter gene expression is monitored using cell culture media or supernatant, the cells can be used for other experiments. Here we show sequential monitoring of forskolin-induced gene expression using the Phospha-Light-EXP SEAP assay to monitor SEAP activity in culture media and cell viability using the Invitrogen PrestoBlue assay on the cells remaining in the culture well. NIH/3T3 cells were transfected with pCRE-SEAP for cAMP-inducible SEAP expression using Lipofectamine LTX reagent with PLUS reagent. Cells were treated with forskolin for 24 hours to induce cAMP production.

Versatility

	Use cells for other applications. Measure SEAP reporter enzyme in samples of culture medium removed from cells. Ideal for performing time course or multiparametric studies -OR-
	Measure SEAP reporter enzyme in the original culture well in the presence of cells, using the same assay protocol and reagent volumes (if cells are not being used for separate assay read-out)
	Measure PLAP (non-secreted) reporter enzyme in cells
	Assay demonstrated with numerous sample types (transiently transfected cell lines, primary cells, stably transfected cell lines, serum from transgenic animals) ^{[4, 7, 12, 13, 15, 17, 18, 21, 22]}See References
	Wide range of applications—gene expression, promoter function, signal transduction pathway activation, gene knockdown, tissue-specific gene therapy targeting, viral function applications such as gene expression, cell-cell fusion, replication, antibody neutralization, infectivity, secretion pathway monitoring, quantitation of SEAP-tagged proteins ^{[1, 8, 9, 10, 11, 14, 19, 20]}See References