不再担心滤光片和二向色性的安装：PHERAstar FSX配备了易于操作、经过优化的光学模块，这些模块包含所有应用特定的滤光片，反光镜，二向色性和/或起偏器，并且可以被酶标仪自动识别。 另外，PHERAstar FSX配备了四个优化的光电倍增管（PMT），可自动选择相应的检测模式。 由于有同时双发射测量模式，您还可以同时进行两个发射波长的测量。
- AlphaScreen®/AlphaLISA®/ AlphaPlex™专用激光光源
NanoBRET™ assay quantitatively evaluates VEGF binding to the VEGFR2 in real-time in living cellsKilpatrick LE1 , Friedman-Ohana R2 , Alcobia DC1 , Riching K2 , Peach CJ1 , Wheal AJ1 , Briddon SJ1 , Robers MB2 , Zimmerman K2 , Machleidt T2, , Wood KV2 , Woolard J1 , Hill SJ1, 1Division of Physiology, Pharmacology and Neuroscience, University of Nottingham, UK , 2Promega Corporation, Madison, WI, USA, 01/2018
Receptor tyrosine kinases (RTK) are transmembrane receptors that translate stimulation by growth factors into regulation of cell growth, proliferation, cell death and differentiation. Consequently, aberrant RTK-signaling is implicated in cancer, making it a popular anti-cancer drug target. Development of pharmaceutical RTK-inhibitors requires assays that characterize receptor-ligand interaction and that identify inhibitors thereof.
A novel method labeled the vascular endothelial growth factor (VEGF) at a single site with the fluorophore TMR. The labeled ligand was used in combination with HEK293 cells expressing the RTK VEGF-receptor 2 (VEGFR2) fused to the NanoLuc® luciferase. Upon interaction of ligand and receptor, bioluminescence resonance energy transfer (BRET) between luciferase and fluorophore takes place.
The signal of luciferase and fluorophore were acquired simultaneously with the PHERAstar microplate reader. This way, not only the pKi of a VEGF-inhibitor was determined, but also the time-course of binding and inhibition was monitored.
CRISPR/Cas9 genome-edited cells express nanoBRET-donor that monitors protein interaction and traffickingCarl White1,2 , Ethan See1,2 , Kevin Pfleger1,2,3, 1Molecular Endocrinology and Pharmacology, Harry Perkins Institute of Medical Research, Australia , 2Centre for Medical Research, The University of Western Australia, Australia , 3Dimerix Limited, Nedlands, Australia, 01/2018
GPCRs are important drug targets requiring receptor-protein interaction and trafficking studies to reveal how they function. Bioluminescence resonance energy transfer (BRET) is a versatile tool to study such interactions and trafficking. Hitherto, it is limited by the ectopic expression of labelled interaction partners. CRISPR/Cas9 genome editing overcomes the limitation by enabling endogenous expression of luciferase-labelled proteins.
CRISPR/Cas9-edited cells endogenously expressing a CXCR4/NanoLuciferase fusion protein were used in conjunction with β-Arrestin/Venus to monitor receptor activation. Employing two fluorophores fused to a membrane and endosome standing CXCR4-interacting protein, respectively, allowed for monitoring of receptor trafficking.
The novel CRISPR/Cas9 technique successfully fused the Nluc BRET donor to endogenously-expressed CXCR4. The resulting protein levels were sufficient to monitor receptor interactions as well as internalization. The internalization assay depends on two acceptor fluorophores whose selective detection was rendered possible by the CLARIOstar’s monochromator.
Assay development for essential enzyme activity in the tegument of live SchistosomesMadhu Sundaraneedi1 , Luke Becker2 , Giovanni Abbenante3 , Alex Loukas2 , Grant Collins1 , Mark Pearson2, 1School of Physical, Environmental and Mathematical Sciences, University of New South Wales, Australia , 2Australian Institute for Tropical Health and Medicine, James Cook University, Australia , 3BMG LABTECH Australia, 12/2017
Schistosomiasis is a parasitic disease that affects over 200 million people in tropical, developing nations, causing severe morbidity and over 300,000 deaths annually. Schistosomiasis is treated with a single drug and no vaccine is available.
We selected three Schistosoma surface-associated enzymes that are indispensable to parasitic survival: alkaline phosphatase; phosphodiesterase SmNPP-5 and an acetylcholinesterase. The activity of these molecules on the surface of live and intact larval and adult Schistosoma can be assayed in real-time of cultured parasites, providing a tool to assess the efficacy of drugs or vaccines targeting these enzymes. The colorimetric assay was read on a FLUOstar® Omega microplate reader.
The versatile instrument supports development of assays and drugs. Apart from its absorbance spectrometer, the FLUOstar Omega is equipped with fluorescence and luminescence detection capabilities allowing fast and reliable endpoint and kinetic measurements in all detection modes.
Fluorescence Polarization based assay for rapid, precise, high-throughput measurement of IgG & Fc containing derivativesDr. Carolanne Doherty, Valitacell, NIBRT, Fosters Avenue, Blackrock, Dublin, Ireland, 11/2017
The accurate, rapid and high-throughput measurement of IgG is essential in the development and manufacture of most therapeutic antibodies. Monoclonal antibodies are becoming increasingly dominant in biopharmaceuticals, where a vast number of samples must be screened for the development of each potential therapeutic.
Here we report the development of a novel, rapid, and simple fluorescence polarization based assay for high-throughput titer measurement of IgG and Fc-containing derivatives. It uses fluorescently labeled protein G that binds IgG. Upon binding, the depolarization of emitted light decreases, this can be detected to report on the presence of IgG. The CLARIOstar®, PHERAstar® and POLARstar® exhibit excellent assay quality when used with the ValitaTITER assay, which enables a high-throughput, simple, precise method for quantification of IgG. Moreover, the assay can be performed using sample straight cell culture supernatant, which means there are no complex sample preparation steps.
Calcium retention capacity assay evaluates inhibition of mitochondrial permeability transition poreM. Awais , D. Latawiec , R. Sutton, Liverpool Pancreatitis Research Group, Institute of Translational Medicine, University of Liverpool, UK, 11/2017
Mitochondrial dysfunction is central to the pathogenesis of acute pancreatitis, ischemia-reperfusion injury of the heart, brain and kidney, muscular dystrophies and neurodegeneration. Mitochondrial dysfunction is the result of a sudden increase in permeability of the inner mitochondrial membrane (IMM), via persistent opening of a multi-protein channel known as the mitochondrial permeability transition pore (MPTP). This is followed by uncontrolled proton flow across the IMM and unregulated flux of water, ions and small solutes into and out of the mitochondrial matrix. This results in rupture of the outer mitochondrial membrane (OMM) and eventually cell death by necrosis. Therefore, MPTP is an attractive target for cell death prevention in a host of disease states.
The calcium retention capacity assay challenges isolated mitochondria with spikes of calcium ions. Upon opening of the MPTP, Ca2+ leaks into the assay buffer and increases fluorescence of the membrane-impermeable CalciumGreen™ dye. The Omega multi-mode plate reader has proven excellent robustness for performing the multiple injections as well as reliable fluorescent detection of the assay.