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donderdag 27 september 2018

Nature Methods Application Notes e-UPDATE: 20 September 2018

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20 September 2018 
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Airyscan detection in multiphoton microscopy: superresolution and improved signal-to-noise ratio beyond the confocal depth limit
https://www.zeiss.com/corporate/int/home.html >
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.

Wheat germ cell-free protein expression enables studies on E3 ubiquitin ligases
http://www.cfsciences.com/eg/ >
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.

Anti-idiotypic binders for Trastuzumab validated in regulatory bioanalysis assay in collaboration with Covance
https://www.moleculardevices.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.

TwistAmp® Liquid: a versatile amplification method to replace PCR
https://www.twistdx.co.uk/ >
Here we introduce TwistAmp® Liquid, a new PCR replacement format that makes RPA technology more amenable to a wide range of research applications. In contrast to PCR amplification, RPA takes minutes, rather than hours, and can be run with little to no equipment. TwistAmp® Liquid Basic and Basic RT kits can be used for applications requiring good fidelity, gel electrophoresis or solid phase detection. TwistAmp® Liquid exo and exo RT allow rapid real-time amplification and detection of targets without sacrificing sensitivity or specificity.

Monitoring neurite morphology and synapse formation in primary neurons for neurotoxicity assessments and drug screening
https://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.

Impact of the Claristep® Filtration System on Recovery and Adsorption of Various Therapeutic Proteins at Low Sample Volumes
https://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.

Rapid and real-time analysis of polyelectrolyte multilayer films using SEEC
http://www.nano-lane.com/ >
Polyelectrolyte multilayers offer novel biosynthetic interfaces that aid in high impact scientific advances in fields such as bioengineering. Current biosensor techniques employed to study the engineered biosynthetic surfaces do not offer real-time visualisation of the layer build-up process and/or surface characterisation studies such as topographic/morphologic analyses. SEEC technology offers enhanced surface contrast that permits quantification of nanometric changes on surfaces such as thin films in liquid conditions. We show that the newly automated N-Lab Station based on SEEC technology offers robust data reproducibility and comprehensive studies including surface characterisation of polyelectrolyte multilayers.


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