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SEAP activity measurement in reporter cell-based assays using BCIP / NBT as substrate

Title data

Jérôme, Valérie ; Freitag, Ruth ; Schüler, Dirk ; Mickoleit, Frank:
SEAP activity measurement in reporter cell-based assays using BCIP / NBT as substrate.
In: Analytical biochemistry. Vol. 585 (2019) . - No. 113402.
ISSN 0003-2697
DOI: https://doi.org/10.1016/j.ab.2019.113402

Abstract in another language

SEAP (secreted embryonic alkaline phosphatase) has been suggested as versatile reporter protein inter alia for cell ligand interaction. Generic photometric assay formats for this enzyme are currently lacking. Using the interaction of recombinant hCD40 ligand with HEK-Blue sensor cells expressing the CD40 receptor as example, we show that such an assay can be developed based on BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium chloride) as substrate. Supplementation of the reaction buffer with a micelle-forming detergent (TWEEN 20) stabilizes the water-insoluble reactions products thereby allowing reproducible photometric quantification of the colloidal dispersion. After optimizing the assay in terms of incubation time, cell number and environmental conditions, a cellular response to stimulation was already visible for 0.25 ng mL−1 of rhCD40L. Moreover, the sensitivity of the assay was significantly better than reported previously for alternative assays used in combination with the commercially available reporter cells. The use of BCIP/NBT as substrate therefore provides a robust and sensitive method to monitor SEAP activity in solution, which could conceivably be extended to other cell-based and biological assays using SEAP as reporter protein.

Further data

Item Type: Article in a journal
Refereed: Yes
Institutions of the University: Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Biology > Chair Microbiology > Chair Microbiology - Univ.-Prof. Dr. Dirk Schüler
Faculties > Faculty of Engineering Science > Chair Process Biotechnology > Chair Process Biotechnology - Univ.-Prof. Dr. Ruth Freitag
Result of work at the UBT: Yes
DDC Subjects: 500 Science > 500 Natural sciences
500 Science > 570 Life sciences, biology
600 Technology, medicine, applied sciences
Date Deposited: 03 Sep 2019 06:18
Last Modified: 03 Sep 2019 06:18
URI: https://eref.uni-bayreuth.de/id/eprint/52126