Nature Methods Application Notes e-UPDATE: 12 August 2014

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

Search/browse Application Notes

12 August 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 One Solution for Cloning and Mutagenesis: In-Fusion®HD Cloning Plus www.clontech.com > 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

		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

		Suitability of Medicyte's upcyte® cells for in vitro formation of liver bud-like organoid liver structures www.medicyte.com > upcyte® cells are non-malignant cell strains derived from primary human cells that underwent targeted genetic modification (upcyte® process) resulting in transiently proliferating cells that can undergo up to 40 population doublings. When contact-inhibited upcyte® cells possess the typical morphology of mature cells and show many of the cell type specific characteristics. Formation of three-dimensional structures resembling embryonic liver buds in vitro has recently been described using stem cell derived cell types (Takebe et al., 2013). Such structures can be transplanted into animals but may also prove useful for ex vivo drug toxicity testing systems to more closely resemble the in vivo situation as compared to conventional two-dimensional (monolayer) cell culture models. In this article we present a protocol using a defined mixture of differentiated human upcyte® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) that form liver buds in vitro which can be cultured for up to 20 days or even longer. These organoid, self-organized liver-like structures contain living cells showing typical functional characteristics of liver cells, e.g. Rifampicin-induced induction of Cytochrome P450 3A4 (Cyp3A4) activity. PDF

		Randomized Adapters for Reducing Bias in Small RNA-Seq Libraries http://www.biooscientific.org > The past decade has seen an explosion of interest in cataloging the small RNA repertoires of animal and plant species, and in understandingthe biological function of small RNAs. Small RNAs include not only microRNAs, but also piRNAs and other types of endogenous small RNAs.Distinguishing closely related small RNAs is difficult using microarray- and qPCR-based approaches, since imperfectly matched small RNAs may still be able to hybridize to PCR primers or immobilized probes. These considerations have led to the realization that next generation sequencing (NGS) is the most practical method for large-scale small RNA studies that aim to identify and enumerate small RNAs in various species and tissues. NGS offers advantages of sensitivity, specificity, and the ability to maximize data acquisition and minimize costs by using multiplex strategies to allow many samples to be sequenced simultaneously. PDF

		How to Choose a Cell Health Assay www.promega.com > Promega has a large portfolio of cell health assays, which has grown significantly since the introduction of the CellTiter 96R Non-Radioactive Cell Proliferation Assay (MTT) in 1991. With so many excellent choices in plate-based assays, choosing the most appropriate one for your experiment can be difficult. Once you have identified the question you are trying to answer, selecting the appropriate assay is much easier. 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

		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 ofTransIT®-LT1, TransIT®-2020 and Lipofectamine® 2000 Transfection Reagents using quantitative reverse transcriptase PCR (RT-qPCR) array and pathway analysis software. 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