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El Granada, CA, United States

Takemoto K.,Hokkaido University | Takemoto K.,Yokohama City University | Matsuda T.,Hokkaido University | McDougall M.,Promega BioSciences | And 7 more authors.
ACS Chemical Biology | Year: 2011

Chromophore-assisted light inactivation (CALI) is a potentially powerful tool for the acute disruption of a target protein inside living cells with high spatiotemporal resolution. This technology, however, has not been widely utilized, mainly because of the lack of an efficient chromophore as the photosensitizing agent for singlet oxygen (1O2) generation and the difficulty of covalently labeling the target protein with the chromophore. Here we choose eosin as the photosensitizing chromophore showing 11-fold more production of 1O2 than fluorescein and about 5-fold efficiency in CALI of â-galactosidase by using an eosin-labeled anti-â-galactosidase antibody compared with the fluorescein-labeled one. To covalently label target protein with eosin, we synthesize a membrane-permeable eosin ligand for HaloTag technology, demonstrating easy labeling and efficient inactivation of HaloTag-fused PKC-ã and aurora B in living cells. These antibody- and HaloTag-based CALI techniques using eosin promise effective biomolecule inactivation that is applicable to many cell biological assays in living cells. © 2011 American Chemical Society. Source

Duellman S.J.,Promega Corporation | Valley M.P.,Promega Corporation | Kotraiah V.,Exonhit Inc. | Vidugiriene J.,Promega Corporation | And 6 more authors.
Analytical Biochemistry | Year: 2013

The aldehyde dehydrogenase (ALDH) family of enzymes is critical for cell survival and adaptation to cellular and environmental stress. These enzymes are of interest as therapeutic targets and as biomarkers of stem cells. This article describes a novel, homogeneous bioluminescence assay to study the activity of the ALDH enzymes. The assay is based on a proluciferin-aldehyde substrate that is recognized and utilized by multiple ALDH enzyme isoforms to generate luciferin. A detection reagent is added to inactivate ALDH and generate light from the luciferin product. The luminescent signal is dependent on the ALDH enzyme concentration and the incubation time in the ALDH reaction; moreover, the luminescent signal generated with the detection reagent is stable for greater than 2 h. This assay provides many advantages over standard NADH fluorescence assays. It is more sensitive and the signal stability provided allows convenient assay setup in batch mode-based high-throughput screens. The assay also shows an accurate pharmacological response for a common ALDH inhibitor and is robust, with a large assay window (S/B = 64) and Z′ = 0.75. © 2012 Elsevier Inc. All rights reserved. Source

Robers M.B.,Promega Corporation | Binkowski B.F.,Promega Corporation | Cong M.,Promega Corporation | Zimprich C.,Promega Corporation | And 8 more authors.
Analytical Biochemistry | Year: 2015

Ligand-mediated endocytosis is a key autoregulatory mechanism governing the duration and intensity of signals emanating from cell surface receptors. Due to the mechanistic complexity of endocytosis and its emerging relevance in disease, simple methods capable of tracking this dynamic process in cells have become increasingly desirable. We have developed a bioluminescent reporter technology for real-time analysis of ligand-mediated receptor endocytosis using genetic fusions of NanoLuc luciferase with various G-protein-coupled receptors (GPCRs). This method is compatible with standard microplate formats, which should decrease work flows for high-throughput screens. This article also describes the application of this technology to endocytosis of epidermal growth factor receptor (EGFR), demonstrating potential applicability of the method beyond GPCRs. © 2015 Elsevier Inc.All rights reserved. Source

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