Nature Methods Application Notes e-UPDATE: 8 April 2014

If you are unable to see the message below, click here to view.

Search/browse Application Notes

8 April 2014  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!

Register to continue receiving application notes updates

FEATURED APPLICATION NOTE Trypsin/Lys-C protease mix for enhanced protein mass spectrometry analysis www.promega.com > Traditionally, trypsin and Lys-C proteases are used in combination to digest proteolytically resistant proteins. Here we describe use of Trypsin/Lys-C Mix for general digestion needs. Working simultaneously under conventional non-denaturing trypsin digestion conditions, Trypsin and Lys-C enhance proteolysis and provide multiple positive effects on protein mass spectrometry analysis including increased number of identified peptides and proteins, higher analytical reproducibility and more accurate protein quantitation. Effectively, by supplementing trypsin with Lys-C, we create improved trypsin. 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

		Molecular indexing for improved RNA-Seq analysis http://www.biooscientific.org > In this application note, Bioo Scientific Corporation and Cellular Research introduce a new kit for high precision gene expression analysis by RNA-Seq. We demonstrate that the NEXTflex™ qRNA-Seq™ Kit can be directly substituted into paired-end library preparation without affecting conventional RNA-Seq analysis methods. This new kit efficiently generates libraries equivalent to standard RNA-Seq libraries with sample barcodes, but with the added feature of molecular indexing. The value of using the added molecular indexing feature depends upon experimental design, data objectives, and sequencing depth for a given library complexity. 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

		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

		Automation of ChIP-Seq Library Preparation for Next Generation Sequencing on the epMotion®5075 TMX http://www.eppendorfna.org > ChIP-Seq library preparation can be a challenging procedure to automate because of its low ChIP DNA input. This Application Note describes the successful automation of Illumina™ ChIP-Seq library preparation on the Eppendorf® epMotion 5075 TMX, using the KAPA High-Throughput Library Preparation Kit from KAPA Biosystems®. Size-selected libraries were prepared from as little as 1 ng of fragmented ChIP DNA. To obtain the recommended 100 ng of library material for sequencing on the Illumina Genome AnalyzerIIx, only 18 amplification cycles were needed for 1 ng of input DNA, or 9 cycles when starting library construction with 10 ng of ChIP DNA. 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

 

Application Notes are supplied by commercial companies. The Nature Publishing Group does not endorse any of the products or services advertised here. You have been sent this Table of Contents Alert because you have opted in to receive it. You can change or discontinue your e-mail alerts at any time, by modifying your preferences on your nature.com account at: www.nature.com/myaccount (You will need to log in to be recognised as a nature.com registrant). For further technical assistance, please contact our registration department For print subscription enquiries, please contact our subscription department For other enquiries, please contact our feedback department Nature Publishing Group | 75 Varick Street, 9th Floor | New York | NY 10013-1917 | USA Nature Publishing Group's worldwide offices: London - Paris - Munich - New Delhi - Tokyo - Melbourne San Diego - San Francisco - Washington - New York - Boston