11779842001

Roche

SEAP Reporter Gene Assay, chemiluminescent

Synonym(s):
secreted alkaline phosphatase assay

Quality Level

usage

sufficient for 250 assays (tubes)
sufficient for 500 assays (microplate)

manufacturer/tradename

Roche

shipped in

wet ice

storage temp.

2-8°C

General description

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.

Specificity

The assay is specifically designed to measure placental AP (alkaline phosphatase), which results in minimal background.

Application

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

Packaging

1 kit containing 5 components.

Specifications

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.

Principle

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.

Other Notes

For life science research only. Not for use in diagnostic procedures.

Kit Components Only

Product No.
Description

  • Alkaline Phosphatase Substrate, CSPD

  • Substrate Buffer, containing Emerald-II luminescence enhancer

  • Inactivation Buffer

  • Dilution Buffer

  • Positive Control, human placental alkaline phosphatase

Pictograms

CorrosionHealth hazard

Signal Word

Danger

Hazard Statements

RIDADR

NONH for all modes of transport

Flash Point(F)

does not flash

Flash Point(C)

does not flash

Travis S Hughes et al.
Molecular therapy : the journal of the American Society of Gene Therapy, 17(1), 88-94 (2008-10-23)
Therapeutic benefit has been reported to result from intrathecal (i.t.) injection of transgene vectors, including naked DNA. However, most studies using naked DNA have measured only the transgene expression of intracellular proteins. Here we demonstrate that i.t. injection of naked...
A synthetic mammalian gene circuit reveals antituberculosis compounds
Weber W, et al.
Proceedings of the National Academy of Sciences of the USA, 105(29), 9994-9998 (2008)

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