Secreted alkaline phosphatase (SEAP) has become a powerful reporter gene for the investigation of promoter activity in transfected eukaryotic cells. The SEAP gene is cloned into vectors behind the promoter under investigation, then transfected into eukaryotic cells. The translated SEAP gene product is secreted from the cells, and is thus easily detected in a sample of culture medium without destroying cells and without time-consuming sample preparation. The elimination of cell lysis permits repeated sampling of the cell medium or other subsequent analysis. The SEAP Reporter Gene Assay, chemiluminescent is based on dioxetane CSPD (chloro-5-substituted adamantyl-1,2-dioxetane phosphate), and provides a convenient and highly sensitive method for the quantitation of transcriptional activity.
The assay is specifically designed to measure placental AP (alkaline phosphatase), which results in minimal background.
SEAP Reporter Gene Assay, chemiluminescent has been used to measure SEAP activity in mouse serum.
The SEAP Reporter Gene Assay is designed specifically for measuring secreted placental AP (alkaline phosphatase) in conditioned cell culture medium from transfected cells. SEAP is secreted into the culture supernatant where it can be easily sampled and assayed. It has been used to measure SEAP activity in cerebrospinal fluid and serum.
Features and Benefits
- Sensitive: Approximately 10fg of alkaline phosphatase
- High dynamic measuring range: Linear range over 4- to 5-orders of magnitude
- Constant light emission: Long-lasting light emission
- Easy to perform: Activity is easily determined from culture medium of the transfected cells
- Safe: No radioactive isotopes
- Fast: Approximately 1 hour
1 kit containing 5 components.
Assay time: approximately 60 minutes
Measuring range: The detection range for alkaline phosphatase is between 10fg and 1ng in a 50μl aliquot. When exceeding 100pg per aliquot, substrate (CSPD) depletion should be considered, as the half-life of the luminescent substrate can be affected.
The chemiluminescent substrate CSPD is dephosphorylated by alkaline phosphatase (AP), resulting in an unstable dioxetane anion that decomposes and emits light. The light emission has maximal activity at a wavelength of 477nm. Chemiluminescence-enhancing reagents, which are found in the substrate buffer, improve the quantum yield of the excited (light-emitting) state more than 500-fold. The light signal, quantified in a tube or microplate luminometer, is linear up to five orders of magnitude and proportional to the concentration of alkaline phosphatase. The signal may also be measured in a scintillation counter (single photon mode). Contaminating alkaline phosphatase activity is eliminated with a heat-inactivation step prior to assaying the reporter gene.
For life science research only. Not for use in diagnostic procedures.