Read by QxMD icon Read

Methods: a Companion to Methods in Enzymology

Paul J Conroy, Ruby H P Law, Tom Caradoc-Davies, James C Whisstock
Antibodies represent a highly successful class of molecules that bind a wide-range of targets in therapeutic-, diagnostic- and research-based applications. The antibody repertoire is composed of the building blocks required to develop an effective adaptive immune response against foreign insults. A number of species have developed novel genetic and structural mechanisms from which they derive these antibody repertoires, however, traditionally antibodies are derived from human, and rodent sources. Due to their high-value therapeutic, diagnostic, biotechnological and research applications, much innovation has resulted in techniques and approaches to isolate novel antibodies...
January 11, 2017: Methods: a Companion to Methods in Enzymology
Philippe Delahaut
Polyclonal antibodies are frequently used as immunodiagnostic tools in fundamental research. They are also used for routine diagnostic purposes in human and veterinary medicine and for quality control procedures in the food-processing industry. The antibody is a major component of the detection system. It binds with the molecule to be identified. This conjugate is subsequently revealed by means of binding the antibody with a radio-isotope, a fluorescent substance, an enzyme inducing a color change, or a biosensor based analytical system...
January 10, 2017: Methods: a Companion to Methods in Enzymology
Daniel Sage, Lauréne Donati, Ferréol Soulez, Denis Fortun, Guillaume Schmit, Arne Seitz, Romain Guiet, Cédric Vonesch, Michael Unser
Images in fluorescence microscopy are inherently blurred due to the limit of diffraction of light. The purpose of deconvolution microscopy is to compensate numerically for this degradation. Deconvolution is widely used to restore fine details of 3D biological samples. Unfortunately, dealing with deconvolution tools is not straightforward. Among others, end users have to select the appropriate algorithm, calibration and parametrization, while potentially facing demanding computational tasks. To make deconvolution more accessible, we have developed a practical platform for deconvolution microscopy called DeconvolutionLab...
January 2, 2017: Methods: a Companion to Methods in Enzymology
Anatole Chessel
This review aims at providing a practical overview of the use of statistical features and associated data science methods in bioimage informatics. To achieve a quantitative link between images and biological concepts, one typically replaces an object coming from an image (a segmented cell or intracellular object, a pattern of expression or localisation, even a whole image) by a vector of numbers. They range from carefully crafted biologically relevant measurements to features learnt through deep neural networks...
January 2, 2017: Methods: a Companion to Methods in Enzymology
Mélodie Duval, Alessandra Marenna, Clément Chevalier, Stefano Marzi
RNA-protein interactions are at the bases of many biological processes, forming either tight and stable functional ribonucleoprotein (RNP) complexes (i.e. the ribosome) or transitory ones, such as the complexes involving RNA chaperone proteins. To localize the sites where a protein interacts on an RNA molecule, a common simple and inexpensive biochemical method is the footprinting technique. The protein leaves its footprint on the RNA acting as a shield to protect the regions of interaction from chemical modification or cleavages obtained with chemical or enzymatic nucleases...
December 24, 2016: Methods: a Companion to Methods in Enzymology
Sushrut Arora, Vikas Saxena, B Vijayalakshmi Ayyar
Antibodies continue to be extremely utilized entities in myriad applications including basic research, imaging, targeted delivery, chromatography, diagnostics, and therapeutics. At production stage, antibodies are generally present in complex matrices and most of their intended applications necessitate purification. Antibody purification has always been a major bottleneck in downstream processing of antibodies, due to the need of high quality products and associated high costs. Over the years, extensive research has focused on finding better purification methodologies to overcome this holdup...
December 22, 2016: Methods: a Companion to Methods in Enzymology
Eric L Van Nostrand, Chelsea Gelboin-Burkhart, Ruth Wang, Gabriel A Pratt, Steven M Blue, Gene W Yeo
Identification of in vivo direct RNA targets for RNA binding proteins (RBPs) provides critical insight into their regulatory activities and mechanisms. Recently, we described a methodology for enhanced crosslinking and immunoprecipitation followed by high-throughput sequencing (eCLIP) using antibodies against endogenous RNA binding proteins. However, in many cases it is desirable to profile targets of an RNA binding protein for which an immunoprecipitation-grade antibody is lacking. Here we describe a scalable method for using CRISPR/Cas9-mediated homologous recombination to insert a peptide tag into the endogenous RNA binding protein locus...
December 18, 2016: Methods: a Companion to Methods in Enzymology
Benedikt M Beckmann
RNA-binding proteins (RBPs) are key players in post-transcriptional regulation of gene expression in eukaryotic cells. To be able to unbiasedly identify RBPs in Saccharomyces cerevisiae, we developed a yeast RNA interactome capture protocol which employs RNA labeling, covalent UV crosslinking of RNA and proteins at 365 nm wavelength (photoactivatable-ribonucleoside-enhanced crosslinking, PAR-CL) and finally purification of the protein-bound mRNA. The method can be easily implemented in common workflows and takes about 3 days to complete...
December 16, 2016: Methods: a Companion to Methods in Enzymology
I F Pinto, C R F Caneira, R R G Soares, N Madaboosi, M R Aires-Barros, J P Conde, A M Azevedo, V Chu
This paper describes microbead-based microfluidic systems. Several aspects of bead assays in microfluidics make them advantageous for bioassays in simple microchannels, including enhanced surface-to-volume ratio, improved molecular recognition reaction efficiency, and the wide range of surface functionalization available with commercial microbeads. Two-level SU-8 molds are used to fabricate PDMS microchannels that can hydrodynamically trap different types of microbeads, with characteristic dimensions of tens of microns...
December 10, 2016: Methods: a Companion to Methods in Enzymology
Sunil K Arya, Pedro Estrela
An immunosensor for the sensitive detection and estimation of tumor necrosis factor-alpha (TNF-α) in undiluted serum has been developed via an electrochemical enzyme-linked immunosorbent assay (ELISA) process. Electrochemical sensing was performed using a TNF-α specific monoclonal antibody modified self-assembled monolayer of dithiobis(succinimidyl propionate) on a comb-shaped gold electrode microarray. After anti-TNF-α antibody binding, unreacted active groups of DTSP were blocked using ethanol amine (EA) and nonspecific binding was prevented using phosphate buffer based starting block T20 (SB)...
December 10, 2016: Methods: a Companion to Methods in Enzymology
Chengqian Che, Tejas Sudharshan Mathai, John Galeotti
This article is a review of registration algorithms for use between ultrasound images (monomodal image-based ultrasound registration). Ultrasound is safe, inexpensive, and real-time, providing many advantages for clinical and scientific use on both humans and animals, but ultrasound images are also notoriously noisy and subject to several unique artifacts/distortions. This paper introduces the topic and unique aspects of ultrasound-to-ultrasound image registration, providing a broad introduction and summary of the literature and the field...
December 10, 2016: Methods: a Companion to Methods in Enzymology
Debashish Ray, Kevin C H Ha, Kate Nie, Hong Zheng, Timothy R Hughes, Quaid D Morris
RNA-binding proteins (RBPs) participate in diverse cellular processes and have important roles in human development and disease. The human genome, and that of many other eukaryotes, encodes hundreds of RBPs that contain canonical sequence-specific RNA-binding domains (RBDs) as well as numerous other unconventional RNA binding proteins (ucRBPs). ucRBPs physically associate with RNA but lack common RBDs. The degree to which these proteins bind RNA, in a sequence specific manner, is unknown. Here, we provide a detailed description of both the laboratory and data processing methods for RNAcompete, a method we have previously used to analyze the RNA binding preferences of hundreds of RBD-containing RBPs, from diverse eukaryotes...
December 10, 2016: Methods: a Companion to Methods in Enzymology
Susan Pérez-Gamarra, Liisa Hattara, Gaurav Batra, Petri Saviranta, Urpo Lamminmäki
Phage display is a well-established and powerful tool for the development of recombinant antibodies. In a standard phage display selection process using a high quality antibody phage library, a large number of unique antibody clones can be generated in short time. However, the pace of the antibody discovery project eventually depends on the methodologies used in the next screening phase to identify the clones with the most promising binding characteristics e.g., in terms of specificity, affinity and epitope...
December 9, 2016: Methods: a Companion to Methods in Enzymology
Trushar R Patel, Grzegorz Chojnowski, Astha, Amit Koul, Sean A McKenna, Janusz M Bujnicki
The diverse functional cellular roles played by ribonucleic acids (RNA) have emphasized the need to develop rapid and accurate methodologies to elucidate the relationship between the structure and function of RNA. Structural biology tools such as X-ray crystallography and Nuclear Magnetic Resonance are highly useful methods to obtain atomic-level resolution models of macromolecules. However, both methods have sample, time, and technical limitations that prevent their application to a number of macromolecules of interest...
December 8, 2016: Methods: a Companion to Methods in Enzymology
Thao Nguyen Le Lam, Claire Morvan, Wenfeng Liu, Chantal Bohn, Yan Jaszczyszyn, Philippe Bouloc
Bacteria optimize their fitness in response to a changing environment by tight regulation of gene expression. Regulation can be controlled at both transcriptional and post-transcriptional levels via key players such as sigma factors, regulatory proteins and regulatory RNAs. The identification of phenotypes associated with gene deletions is the established method for finding gene functions but may require testing many conditions for each studied mutant. As regulatory RNAs often contribute to fine-tuning gene expression, phenotypes associated with their inactivation are often weak and difficult to detect...
December 2, 2016: Methods: a Companion to Methods in Enzymology
Jean-François Gilles, Marc Dos Santos, Thomas Boudier, Susanne Bolte, Nicolas Heck
We present a new plugin for ImageJ called DiAna, for Distance Analysis, which comes with a user-friendly interface. DiAna proposes robust and accurate 3D segmentation for object extraction. The plugin performs automated object-based co-localization and distance analysis. DiAna offers an in-depth analysis of co-localization between objects and retrieves 3D measurements including co-localizing volumes and surfaces of contact. It also computes the distribution of distances between objects in 3D. With DiAna, we furthermore introduce an original method, which allows for estimating the statistical significance of object co-localization...
November 24, 2016: Methods: a Companion to Methods in Enzymology
Hanchao Zhao, Susan A Martinis
Aminoacyl-tRNA synthetases (AARSs) comprise an enzyme family that generates and maintains pools of aminoacylated tRNAs, which serve as essential substrates for protein synthesis. Many protein synthesis factors, including tRNA and AARSs also have non-canonical functions. Particularly in mammalian cells, alternate functions of AARSs have been associated with re-distribution in the cell to sites that are removed from translation. Sub-fractionation methods for E. coli were designed and optimized to carefully investigate re-localization of bacterial AARSs and tRNA that might aid in conferring alternate activities...
November 23, 2016: Methods: a Companion to Methods in Enzymology
Pengfei Fang, Min Guo
Aminoacyl-tRNA synthetases (aaRSs) are enzymes that function at the first step of translation, catalyzing the conjugation of amino acids to their cognate tRNAs for protein synthesis. While preserving this essential role, higher eukaryotic aaRSs, such as human cytoplasmic aaRSs, have developed other functions during evolution, including angiogenesis, inflammation, development, tumorigenesis, etc. These translational and nontranslational functions of aaRSs are attractive targets for developing antibacterial, antifungal, anticancer agents and for treating other human diseases...
November 22, 2016: Methods: a Companion to Methods in Enzymology
Stephanie N Oprescu, Laurie B Griffin, Asim A Beg, Anthony Antonellis
Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed, essential enzymes responsible for charging tRNA with cognate amino acids-the first step in protein synthesis. ARSs are required for protein translation in the cytoplasm and mitochondria of all cells. Surprisingly, mutations in 28 of the 37 nuclear-encoded human ARS genes have been linked to a variety of recessive and dominant tissue-specific disorders. Current data indicate that impaired enzyme function is a robust predictor of the pathogenicity of ARS mutations...
November 20, 2016: Methods: a Companion to Methods in Enzymology
Min Shen, James F Rusling, Chandra K Dixit
Immobilized antibody systems are the key to develop efficient diagnostics and separations tools. In the last decade, developments in the field of biomolecular engineering and crosslinker chemistry have greatly influenced the development of this field. With all these new approaches at our disposal, several new immobilization methods have been created to address the main challenges associated with immobilized antibodies. Few of these challenges that we have discussed in this review are mainly associated to the site-specific immobilization, appropriate orientation, and activity retention...
November 19, 2016: Methods: a Companion to Methods in Enzymology
Fetch more papers »
Fetching more papers... Fetching...
Read by QxMD. Sign in or create an account to discover new knowledge that matter to you.
Remove bar
Read by QxMD icon Read

Search Tips

Use Boolean operators: AND/OR

diabetic AND foot
diabetes OR diabetic

Exclude a word using the 'minus' sign

Virchow -triad

Use Parentheses

water AND (cup OR glass)

Add an asterisk (*) at end of a word to include word stems

Neuro* will search for Neurology, Neuroscientist, Neurological, and so on

Use quotes to search for an exact phrase

"primary prevention of cancer"
(heart or cardiac or cardio*) AND arrest -"American Heart Association"