Nature Methods Application Notes e-UPDATE: 9 October 2012

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9 OCTOBER 2012  Application Notes bring you the latest information about innovative tools and technologies and their applications in the lab. We hope that you will find this service useful and informative and encourage you to sign up for future Updates to ensure that you never miss one!

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FEATURED APPLICATION NOTE Long-span, mate-pair scaffolding and other methods for faster next-generation sequencing library creation www.lucigen.com > The NxSeq™ 40 kb Mate-Pair Cloning Kit facilitates the creation of scaffolds for de novo genome assembly. Supporting either Illumina or 454 sequencing, the kit produces long-span, mate-pair sequences with greater efficiency than existing protocols. In addition, NxSeq DNA Sample Prep Kits can be used to streamline workflow and speed up DNA library preparation for next-generation sequencing. PDF

		High-content analysis of biomarker intensity and distribution in 3D microtissues www.perkinelmer.com > Three-dimensional (3D) cell culture methods are widely accepted as more physiologically relevant than conventional 2D cell culture methods and are believed to improve the prediction of drug candidates at an early stage of the drug development process. Here we describe the analysis of a spherical colon cancer microtissue model using the Operetta® High Content Imaging System. In vivo near-infrared (NIR) agents allowed visualization and quantification of cancer-associated biomarker intensity and distribution in microtissues. PDF

		Understanding the impact of cancer cells' metabolic phenotype and intervention strategies www.seahorsebio.com > Cancer cells exhibit high rates of glycolysis, even in the presence of oxygen, a phenomenon known as the Warburg effect. While normal cells generate most of their ATP via oxidative phosphorylation, cancer cells are more reliant on glycolysis to generate ATP. Energy production through glycolysis results in a net production of protons; as glycolysis occurs, protons are extruded into the extracellular environment, acidifying the medium surrounding the cells or tissue. PDF

		ProDM™ – A kit for tryptic digestion monitoring for a successful shotgun proteomics www.itsibio.com > Successful shotgun proteomics depends on optimal digestion of proteins into peptides prior to LC/MS/MS. Current methods to determine the status of protein digestion are time consuming and/or require expensive reagents and equipment. We describe a method for determining the extent of protein digestion using ProDM™ kit and a basic spectrophotometer. Determining whether the protein is digested or not prior to LC/MS/MS will avoid wasting instrument time, samples and money. PDF

		Qualitative and Quantitative Characterization of the Metabolome, Lipidome, and Proteome of Human Hepatocytes www.waters.com > A label-free multi-omics approach has been applied for the analysis of the transfected human hepatocyte cells by implementing LC/HDMS[E], providing both qualitative and quantitative information within a single experiment. PDF

		A rapid, directional RNA-seq library preparation workflow for Illumina® sequencing www.epibio.com > Most current RNA-seq library preparation methods are time-consuming, multistep processes. We describe a workflow that includes the Ribo-Zero™ and ScriptSeq™ v2 Kits that enables researchers to go from total RNA to cluster-ready RNA-seq libraries in less than 1 d. The RNA-seq libraries produced are virtually free of contaminating ribosomal RNA (rRNA) and provide for directional paired-end and multiplex sequencing on Illumina® sequencing platforms. PDF

		LentiBrite™ Lentiviral Biosensors for Fluorescent Cellular Imaging: Analysis of Autophagosome Formation www.millipore.com > Expression of genetically-encoded fluorescently-tagged proteins has widely been employed for real-time visualization of cellular behavior and trafficking. Prepackaged, ready-to-use, high-titer lentiviral particles (which we have termed "lentiviral biosensors") encoding GFP- or RFP-tagged proteins are a convenient, robust solution for fluorescent imaging of transduced cells. Compared to other nonviral transfection methods, lentiviral transduction, in many cases, offers higher transfection efficiency and more homogeneous protein expression, particularly for traditionally hard-to-transfect primary cell types. Lentiviral biosensors are ideal for use with fixed and live cell fluorescent microscopy, and are non-disruptive towards cellular function. GFP- or RFP-protein localization matches well with antibody-based immunostaining and demonstrates altered patterns of expression upon treatment with modulators of cell function and phenotype. Lentiviral biosensors provide a broadly effective, convenient method for visualization of cell behavior under a variety of physiological and pathological treatment conditions, in both endpoint and real-time imaging modalities. In this study, we focus on lentiviral biosensors containing GFP-LC3 and RFP-LC3 for the study of autophagosome formation. PDF

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