Nature Methods Application Notes e-UPDATE: 13 May 2014

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FEATURED APPLICATION NOTE Measuring Intracellular Protein Lifetime Dynamics Using NanoLuc® Luciferase www.promega.com > Adaptive stress response pathways are tightly regulated by the localization and concentration of key intracellular transcription factors. The rapid changes in protein lifetime that occur following environmental or chemical stress can be exploited using a new genetic reporter strategy that includes NanoLuc® luciferase. NanoLuc® luciferase is ideally suited as a protein function reporter for measuring changes in transcription factor levels following induction of stress response signaling. Via genetic attachment of NanoLuc® luciferase to various target proteins, induction of these pathways can be measured using a simple, luminescent assay readout. The physical properties of NanoLuc® luciferase (e.g., small size, ATP-independence, extreme brightness, etc.) make an excellent reporter for quantifying endogenous levels of these naturally low-abundance proteins. NanoLuc® luciferase-enabled assays for monitoring the intracellular levels of these highly dynamic proteins provides an excellent complement to existing reporter gene assays for toxicological screening and general pathway analysis. PDF

		One Solution for Cloning and Mutagenesis: In-Fusion®HD Cloning Plus www.clontech.com/takara > In this application note, we present a single, convenient system for both cloning and site-directed mutagenesis including deletions, base substitutions, or insertions. In-Fusion HD Cloning Plus is sequence independent, seamless, directional, flexible enough to use with any vector and allows over 95% cloning efficiency to be consistently achieved. Mutagenesis can be carried out by combining the strength of the In-Fusion®HD with inverse PCR. PDF

		Streamlined Library Construction for Quantitative, Directional, and Standard RNA-Seq http://www.biooscientific.org > RNA Sequencing (RNA-Seq) is a valuable tool for a broad range of clinical, environmental, and basic research. Producing high quality RNA-Seq libraries can be challenging for several reasons, including isolation of pure RNA, efficiently converting RNA to cDNA, and loss of material incurred during the series of enzymatic steps and cleanups required for library construction. Here we introduce Bioo Scientic's family of Illumina compatible NEXTflex™ Rapid RNA-Seq kits, all of which include the thermostable NEXTflex™ Rapid Reverse Transcriptase enzyme. The NEXTflex™ Rapid RNA-Seq Kits provide affordable and unique technology, some of the shortest RNA-Seq library preparation times of any kits on the market, include all enzymes required for library preparation, and produce the highest quality RNA-Seq library. PDF

		The Impact of Transfection Mediated Toxicity - Gene Expression and Cytotoxicity Analysis of Transfection Reagents www.mirusbio.com > While plasmid DNA delivery is a widely used method to study cellular functions of proteins of interest, studies to identify nontoxic gene delivery reagents are limited. With the advent of high-information content technologies, especially RT-qPCR array, it is now possible to identify the gene expression response to a particular cellular insult. This improvement, coupled with the observation that virtually all toxic responses are accompanied by changes in gene expression, suggests that gene expression analysis has the potential to refine the identification of minimal‐toxicity transfection reagent where phenotypic responses such as altered morphology is not immediately evident. Consequently, we conducted an integrative study to explore the conventional toxicological endpoints and to identify the minimal-transcriptomic effects of TransIT®-LT1, TransIT®-2020 and Lipofectamine® 2000 Transfection Reagents using quantitative reverse transcriptase PCR (RT-qPCR) array and pathway analysis software. PDF

		Chemiluminescent Westerns: How film and photochemistry affect experimental results www.licor.com > Since the 1970s, enhanced chemiluminescence has been used to detect proteins on Western blots. Secondary antibodies are labeled with the horseradish peroxidase (HRP) enzyme, which oxidizes the luminol-based chemiluminescent substrate and causes it to transiently produce light at ~428 nm. This sensitive, reliable detection chemistry is typically documented by exposure of the blot to x-ray film, which darkens in response to the emitted light. Signal intensity is determined by the number of HRP molecules reacting with substrate. Chemiluminescent blots are often analyzed by visual assessment of band intensities. This method is sufficient to confirm presence or absence of a signal, or to compare bands of substantially different intensities. For more detailed analysis, film images may be digitized and relative band intensifies measured by densitometry. PDF

		High-content analysis of three-dimensional tumor spheroids: investigating signaling pathways using small hairpin RNA www.3dbiomatrix.com > www.ttplabtech.com > http://www.gnf.org > In the past, creating three-dimensional (3D) tumor spheroids that were suitable for high-throughput screening (HTS) was a difficult and often expensive process. We describe how to couple easy, controllable 3D spheroid formation in Perfecta3D® Hanging Drop Plates with the acumen® eX3 high-content imager for rapid, multicolor, whole-well quantification. This process provides researchers with a highly efficient method to achieve physiologically relevant tumor models in an HTS format. PDF

		Rapid ELISA-Based Measurement of Protein Phosphorylation Using RayBio® Phosphorylation ELISA Kits http://www.raybiotech.org > RayBio® Phosphorylation ELISA is a rapid, convenient and sensitive sandwich ELISA for the in vitro measurement of key signaling pathway phosphoproteins in cell or tissue extracts. Over 20 different kits are available for well-studied pathway molecules such as EGFR, Akt, Erk1/2, p38 alpha, Mek1, STAT1, STAT3, and Met. RayBio Phosphorylation ELISA also features site-specific phospho-antibodies which can detect a single phosphorylated residue (for example, Tyr1086 of EGFR). This method is an improvement on traditional labor-intensive immunoblotting protocols that require 2-3 days of processing time. PDF

 

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