Tuesday, August 14, 2018

Nature Methods Application Notes e-UPDATE: 13 August 2018

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13 August 2018 
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Monitor NF-κB activation with a sensitive dual reporter assay on the SpectraMax iD5 microplate reader
https://www.moleculardevices.com/ >
Reporter genes are valuable tools for studying gene expression, serving as surrogates for genes involved in various signaling pathways and disease conditions. Luciferases are the most common reporter genes, as they are easily detectable using luminometers or luminescence microplate readers, and the low luminescent background present in cells enables high assay sensitivity. Firefly luciferase is typically used as a readout for expression of a gene of interest. Luciferase from the sea pansy Renilla reniformis is often used in multiplexed luciferase assays as a second reporter to normalize for transfection efficiency, cell viability, and other factors that may vary among assay samples.

Controlling the topography and biochemistry of cell culture substrates with PRIMO® photopatterning system
https://www.alveolelab.com >
In vivo, the cellular microenvironment has a crucial impact on the regulation of cell behavior and functions such as cellular differentiation, proliferation and migration. One of the challenges confronting cell biologists is to mimic this microenvironment in vivo to more efficiently study living cells and model diseases. Here we present PRIMO: a contactless and maskless UV projection system, which allows to control the topography and chemistry of in vivo microenvironments. We first show that PRIMO is a suitable tool to structure photosensitive resists and create molds on which elastomeric solutions (PDMS) can be polymerized. Then we show that the structured PDMS can be specifically functionalized with biomolecules using UV-light structured by PRIMO and a specific photo-initiator (PLPP™). Altogether, we demonstrate that PRIMO allows to tailor the cell microenvironment topography through microfabrication and biochemistry through micropatterning.

Anti-idiotypic binders for Trastuzumab validated in regulatory bioanalysis assay in collaboration with Covance
www.avacta.com/ >
Regulatory bioanalysis of therapeutic proteins during drug development and clinical follow-up requires the use of critical reagents that can specifically identify and accurately quantify each biotherapeutic within patient samples. Antibodies currently represent the 'gold standard' of affinity reagents used in the regulated bioanalysis of therapeutic proteins. While traditional antibodies have been refined to the point where they are specific, sensitive and reasonably reliable, they can be limited by their development speed, complexity to produce on an industrial scale and lot-to-lot variation in assay performance.

Quantitative live-cell analysis for optimization of culture conditions and evaluation of cell health in human induced pluripotent stem cell-derived neurons
www.essenbioscience.com/en/ >
A major limitation in studying human diseases affecting the nervous system is the ability to culture, monitor and analyze neuronal cells that accurately represent human phenotypes of these disorders. The use of human induced pluripotent stem cell (hiPSC)-derived neurons has provided a new approach aimed at modeling neurological diseases. Monitoring neuronal morphology and cell health in long-term culture is critical for the characterization and evaluation of these novel model systems. Traditional approaches rely on endpoint assays and imaging techniques that require immunochemical staining, which lacks real-time kinetic information.

Impact of the Claristep® Filtration System on Recovery and Adsorption of Various Therapeutic Proteins at Low Sample Volumes
www.sartorius.com/sartoclear-dynamics-lab >
We tested the novel filter device Claristep® for the preparation of protein samples containing one of four different target molecules, i.e. an anti-malaria vaccine candidate, aviscuminum, interferon alfa-2B or monoclonal antibody (mAb) P2G12. In comparison to the standard protocols for sample preparation prior to analysis by either reversed-phase or SEC HPLC, the Claristep® filter did not exhibit a significant difference in protein concentration if the target molecules were present with at least 1.0 g L-1. A significantly reduced product concentration was observed in the Claristep® filtered samples compared to the standard protocol for mAb P2G12 if the concentrations were lower. The protein adsorption to the filter material was well described by a Langmuir adsorption isotherm. Besides, the Claristep® filters facilitated a rapid sample preparation with minimal volume losses.

Monitoring neurite morphology and synapse formation in primary neurons for neurotoxicity assessments and drug screening
www.thermofisher.com/in/en/home.html >
Synapse formation during nervous system development and degeneration in the pathogenesis of human neurological diseases are highly regulated processes. Subtle changes in the environment of the complex neuronal network may cause either breakdown or creation of synaptic connections. Drug discovery screening for neurological diseases and compound neurotoxicity evaluation would benefit from robust, automated, quantitative in vitro assays that monitor neuronal function. We hypothesized that (1) toxic insults to the nervous system will cause neuronal synapses to deteriorate in the early phase of neurotoxicity, eventually leading to neurite degeneration and neuronal cell death if the damage is severe; and (2) an in vitro functional assay for synapse formation and neuronal morphology could be used to monitor and identify such neurotoxic events. We thus developed an automated, functional, high-content screening (HCS) imaging assay to track and quantify the dynamic changes in neurites and synapses. This assay facilitates automation and streamlining of a laborious process in drug discovery screening and compound neurotoxicity assessment. The assay also enables quantitative comparisons between compounds that affect neuronal morphology and function, particularly in neuriteand synapse-associated events.

Wheat germ cell-free protein expression enables studies on E3 ubiquitin ligases
http://cfsciences.com/eg/index.html >
With about 600 to 700 E3 ubiquitin ligases encoded within the human genome, it becomes a complex task to map out the substrate-E3 ligase interactions underlying the regulation of protein degradation in the cell. The wheat germ cell-free protein expression system from CFS, however, provides suitable means for preparing E3 ligases on a high throughout. This allowed different groups to do protein-protein interaction screening experiments and to identify binding partners for E3 ligases.


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