Quantification of gene expression with a secreted alkaline phosphatase reporter system

BioTechniques
T T YangS R Kain

Abstract

The cDNA encoding secreted alkaline phosphatase (SEAP) is a useful tool for investigating the function of known or putative enhancer/promoter elements. SEAP has the unusual properties of extreme heat stability and resistance to the phosphatase inhibitor L-homoarginine. Therefore, endogenous alkaline phosphatase activity in transfected cells can be minimized by pretreatment of samples at 65 degrees C and incubation with the inhibitor. With the use of the chemiluminescent substrate CSPD, 10(-13) g of enzyme can be detected in culture medium, and the enzyme activity can be detected as early as 24 h after transfection. The chemiluminescence-based SEAP assay is about 10-fold more sensitive than similar assays using firefly luciferase as the reporter enzyme. The SEAP activity can also be assayed with a fluorescent substrate MUP, which provides sensitivity comparable to luciferase. Since the enzyme is secreted to culture medium, the enzyme assay can be performed on small samples of the culture supernatant. Because preparation of cell lysates is not required, assaying for SEAP activity is faster and more convenient than assaying for intracellular reporters. Furthermore, because the transfected cells are not disturbed by the sampling pr...Continue Reading

Citations

Sep 11, 2004·Archives of Pharmacal Research·Xing Fu CaiYoung Ho Kim
Oct 12, 2004·Phytotherapy Research : PTR·Xing Fu CaiYoung Ho Kim
Jan 6, 2006·Archives of Pharmacal Research·Phan Van KiemYoung Ho Kim
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Oct 29, 2013·The Biochemical Journal·Tal GlinkaAriel Stanhill
Oct 6, 2004·Archives of Pharmacal Research·Xing Fu CaiYoung Ho Kim
Sep 24, 2004·The Journal of Gene Medicine·Ibrahim ElmadbouhJean-Baptiste Michel
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