Miniaturization and improved throughput of the BCA concentration determination methodCarl Peters, BMG LABTECH, USA, 05/2018
Determining total protein concentration is an accepted means of comparing and standardizing biological samples. The utility of protein concentration assessment extends to high-throughput screening platforms including proteomic and genomic applications. Ideally, the protein concentration determination should also be amenable to high throughput. Most protein determination assays use absorbance measurement detection which is difficult to minimize for higher throughput.
Previously, it has been reported that colorimetric assays, including the bicinchoninic acid (BCA) protein assay, can be performed in white plates using fluorescence detection. The method exploits the inherent fluorescence of white plates. In presence of an absorbing solution, the inherent fluorescence is quenched and the decrease in fluorescent signal can be used to measure colorimetric assays. This principle was employed to miniaturize the BCA protein quantification assay down to 384- and 1536- well plates.
PHERAstar measures AlphaScreen assay to develop selective inhibitors for the human YEATS domainsThomas Christott , Carmen Coxon , James Bennett , Charline Giroud , Octovia Monteiro , Oleg Fedorov , , Structural Genomics Consortium, University of Oxford, UK, 04/2018
YEATS domains are epigenetic regulators and are recognised as readers of histone post-translational modifications (HPTM) alongside bromodomains, PHD fingers, and others. YEATS domains bind to lysine when the ε-carbon is acetylated or crotonylated. The YEATS-containing ENL links histone acetylation to active transcription and is a major driver of several types of acute leukaemia. Hence, ENL is a rational drug target to attenuate aberrant cell growth and malignancy.
An AlphaScreen assay that reports on the interaction of modified histone 3 with the YEATS domains of different proteins such as ENL was developed. The inhibitor screening was performed on a PHERAstar® microplate reader and uncovered a potent small molecule inhibitor interfering with the YEATS domains of ENL and AF9.
The PHERAstar FSX provided a powerful and versatile platform for the drug discovery campaign. Here, the inhibitor screening of tens of thousands of compounds for inhibiting the YEATS domain with an AlphaScreen® approach resulted in the identification of a potent small molecule inhibitor.
Cell-based assay detects residual β-blocker substances in effluent of municipal wastewater treatment plantsK. Bernhard , C. Stahl , R. Martens , M. Frey, SIZ Zellkulturtechnik, c/o Hochschule Mannheim, Mannheim, Germany, 03/2018
Residues of human pharmaceuticals, such as β-blockers are increasingly found in effluent wastewater of treatment plants (WWTP) and represent a potential environmental threat. Beta-blockers antagonize β-adrenergic receptors and thereby control hypertension and cardiac arrhythmias. The amino acid sequence of the target, the β1-adrenergic receptor, is evolutionarily highly conserved among vertebrates. Thus, organisms may physiologically respond to β-blockers. For environmental risk assessment one has to know the extent to which aquatic organisms are exposed to β-blockers and metabolites with the same mode of action (MOA).
The mode of action-based β-blocker assay in living cells measures total β-blocker activities in complex mixtures such as WWTP effluents as equivalents of the lead substance metoprolol (MetEQ). The assay is suitable as a standard method in large-scale monitoring of WWTP effluents and the aquatic environment they are discharged into. The CLARIOstar® microplate reader provides highly stable and reliable results as well as the required robustness for continuous use.
FRET sensor assesses ATP levels in living plants experiencing low oxygen conditionsStephan Wagner1,2 , Philippe Fuchs1,2 , Thomas Nietzel1,2 , Marlene Elsässer1,2 , Markus Schwarzländer1,2, 1 Institute of Plant Biology and Biotechnology, University of Münster, Germany , 2 Institute of Crop Science and Resource Conservation (INRES), University of Bonn, Germany, 02/2018
To survive as sessile organisms, plants need to constantly adapt their metabolism to their environment. Flooding of plants is widespread and has severe metabolic consequences as it limits the cellular supply with oxygen to drive respiration, and thus production of ATP. Submergence acclimation typically involves drastic alterations in metabolism to circumvent anoxia and to maintain primary metabolism and ATP supply. We have recently established the use of a genetically encoded protein sensor (ATeam1.03-nD/nA) to assess MgATP dynamics in the model plant Arabidopsis thaliana.
This FRET-system read on a CLARIOstar® allows for reliable analyses of changes in MgATP in vivo and in real-time and provides a new handle to investigate the energetic consequences of low oxygen for the plant cell. The microplate reader’s orbital averaging function acquires the signal in multiple points of the well to obtain a meaningful result of the non-homogenously distributed Arabidopsis seedlings.
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.