Advanced Search

Journal Navigation

Journal Home

Subscriptions

Archive

Contact Us

Table of Contents

Click here for more information

Sign In to gain access to subscriptions and/or personal tools.
Journal of Biomolecular Screening
This Article
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
1087057107301497v1
12/5/694    most recent
Right arrow References
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to Saved Citations
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Request Reprints
Right arrow Add to My Marked Citations
Citing Articles
Right arrow Citing Articles via Web of Science (7)
Right arrow Citing Articles via Google Scholar
Right arrow Citing Articles via Scopus
Google Scholar
Right arrow Articles by Bovolenta, S.
Right arrow Articles by Corazza, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bovolenta, S.
Right arrow Articles by Corazza, S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Development of a Ca2+-Activated Photoprotein, Photina®, and Its Application to High-Throughput Screening

Silvia Bovolenta

Axxam SpA, San Raffaele Biomedical Science Park, Milan, Italy

Maria Foti

Department of Biotechnology and Bioscience, University of Milano-Bicocca, Milan, Italy

Stefan Lohmer

Axxam SpA, San Raffaele Biomedical Science Park, Milan, Italy

Sabrina Corazza

Axxam SpA, San Raffaele Biomedical Science Park, Milan, Italy, sabrina.corazza.sc{at}axxam.com

The present work describes the engineering and characterization of a new Ca2+-activated photoprotein (Photina®) and its use in mammalian cell lines for implementation of flash luminescence cell-based assays for high-throughput screening (HTS). When used to measure the activation of 2 G protein—coupled receptors (GPCRs), targeting Photina® to the mitochondria increased the signal strength as compared to the normal cytoplasmic expression of Photina.® The mitochondrial-targeted Photina® also produced a higher signal-to-noise ratio than conventional calcium dyes and a consistently stronger signal than aequorin when tested under equivalent conditions. MitoPhotina® provided strong and reliable results when used to measure the activity of purinergic receptors endogenously expressed in the Chinese Hamster Ovary cells and heterologously expressed GPCRs in response to their cognate ligands. Several different types of flash luminescence plate readers (FLIPR3, FLIPRTETRA, CyBi®-®Lumax flash HT, Lumilux®, Lumibox) in different plate formats (96, 384, 1536 wells) were used to validate the use of Photina in HTS. The cell number had to be adjusted to correspond to the qualities of the different readers, but once so adjusted, it provided equivalent results on each device. The results obtained show robust and reproducible light signals that offer new possibilities for application of photoproteins to the generation of cell-based assays for HTS. (Journal of Biomolecular Screening 2007:694-704)

Key Words: Photina® • photoprotein • flash luminescence • HTS • cell-based assays • GPCR

This version was published on August 1, 2007

Journal of Biomolecular Screening, Vol. 12, No. 5, 694-704 (2007)
DOI: 10.1177/1087057107301497
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?