Introduction
Secrete-Pair™ Gaussia Luciferase Dual and Single Luminescence Assay Kits
GeneCopoeia’s Secrete-Pair™ Dual Luminescence Assay Kit is designed to analyze the activities of Gaussia Luciferase (GLuc) and Secreted Alkaline Phosphatase (SEAP) in a dual-reporter system. Both GLuc and SEAP are secreted reporter proteins, permitting detection without cell lysis. Secrete-Pair measures dual reporter signals and allows transfection normalization. A single GLuc reporter system is also available.
Advantages
• Live cell assays. Secreted GLuc and SEAP make cell lysis unnecessary. The same sample can be assayed multiple times for different time points, environmental conditions, etc., reducing sample-to-sample variation. Ideal for high-throughput applications.
• Dual-reporter detection. SEAP allows normalization of GLuc signal for greater accuracy.
• Sensitive and robust system. GLuc is 1,000 times more sensitive than firefly and Renilla luciferases.
• Flexible assay conditions. Two robust buffer conditions are provided for GLuc assays: 1) Buffer for greater stability retains more than 90% of signal within the first 10 minutes, extending the half-life of luminescence to 30 minutes; 2) Buffer for higher sensitivity for detecting low GLuc expression.
• Clone and vector compatibility. Compatible with GeneCopoeia’s GLuc-ON™ promoter reporter clones, miTarget™ miRNA target clones, GLuc-ON™ Transcriptional Response Element (TRE) clones, and cloning vectors.
Protocol and Data
Protocol Overview
Figure 1. General workflow for the Secrete-Pair™ Dual Luminesence Assay System.
Dual-reporter system
The Secrete-Pair™ Dual Luminescence Assay System is ideally suited for dual-reporter vectors and clones, such as those available for the GLuc-ON™ promoter clones and miTarget™ microRNA target clones. The secondary reporter, secreted Alkaline Phosphatase (SEAP), serves as an internal control. The dual-reporter system enables transfection normalization for accurate cross-sample comparison (Figure 2).
| Figure 2. Normalized promoter activities in H1B1B and HEK293T cells. Dual-reporter promoter clones or controls were transfected into two cell lines in duplicates. Samples were analyzed 24 hrs (HEK293T) and 48 hrs (H1B1B) after transfection. NEG (containing non-promoter sequence) and EMPTY (no promoter in the vector) are negative controls. |
Two buffer conditions are provided in the kit for GLuc assays depending on the application. Buffer GL-S contains a stabilizer and can be used for stabilized activity by overcoming the quick decay of the GLuc signal. When higher signal intensities are needed, Buffer GL-H is used instead (Figure 3).
Figure 3. GLuc and SEAP assays. Cell culture medium was collected from cells transfected with wild-type (wt) GLuc- SEAP dual-reporter clone. 10 μl of the medium was used in each assay. At the beginning, the GLuc activity in Buffer GL- H is about 4-6 times higher than that in Buffer GL-S, then quickly decays. The GLuc activity in Buffer GL-S, however, is much more stable.
