• Volume 8, 2018
  • Volume 7, 2017
  • Volume 6, 2016
  • Volume 5, 2015
  • Volume 4, 2014
  • Volume 3, 2013
  • Volume 2, 2012
  • Volume 1, 2011

Biochemistry

Hydrogen Deuterium Exchange Mass Spectrometry of Oxygen Sensitive Proteins Authors:  Luke Berry, Angela Patterson, Natasha Pence, John W. Peters and Brian Bothner, date: 03/20/2018, view: 2035, Q&A: 0
The protocol detailed here describes a way to perform hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) on oxygen sensitive proteins. HDX-MS is a powerful tool for studying the protein structure-function relationship. Applying this technique to anaerobic proteins provides insight into the mechanism of proteins that perform oxygen ...
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Cancer Biology

Detection and Analysis of Circular RNAs by RT-PCR Authors:  Amaresh C Panda and Myriam Gorospe, date: 03/20/2018, view: 4143, Q&A: 2
Gene expression in eukaryotic cells is tightly regulated at the transcriptional and posttranscriptional levels. Posttranscriptional processes, including pre-mRNA splicing, mRNA export, mRNA turnover, and mRNA translation, are controlled by RNA-binding proteins (RBPs) and noncoding (nc)RNAs. The vast family of ncRNAs comprises diverse regulatory ...
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Developmental Biology

Quantitative Live-cell Reporter Assay for Noncanonical Wnt Activity Authors:  Edith P. Karuna, Michael W. Susman and Hsin-Yi Henry Ho, date: 03/20/2018, view: 1910, Q&A: 0
Noncanonical Wnt signaling functions independently of the β-catenin pathway to control diverse developmental processes, and dysfunction of the pathway contributes to a number of human pathological conditions, including birth defects and metastatic cancer. Progress in the field, however, has been hampered by the scarcity of functional assays for ...
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Ciliary Assembly/Disassembly Assay in Non-transformed Cell Lines Authors:  Masaki Saito, Kensuke Sakaji, Wataru Otsu and Ching-Hwa Sung, date: 03/20/2018, view: 1937, Q&A: 0
The primary cilium is a non-motile sensory organelle whose assembly and disassembly are closely associated with cell cycle progression. The primary cilium is elongated from the basal body in quiescent cells and is resorbed as the cells re-enter the cell cycle. Dysregulation of ciliary dynamics has been linked with ciliopathies and other human ...
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Measurement of Intracellular ROS in Caenorhabditis elegans Using 2’,7’-Dichlorodihydrofluorescein Diacetate Authors:  Dong Suk Yoon, Myon-Hee Lee and Dong Seok Cha, date: 03/20/2018, view: 2422, Q&A: 1
Reactive oxygen species (ROS) are generated during normal metabolic processes under aerobic conditions. Since ROS production initiates harmful radical chain reactions on cellular macromolecules, including lipid peroxidation, DNA mutation, and protein denaturation, it has been implicated in a wide spectrum of diseases such as cancer, ...
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Microbiology

Bacterial Cell Wall Precursor Phosphatase Assays Using Thin-layer Chromatography (TLC) and High Pressure Liquid Chromatography (HPLC) Authors:  Manuel Pazos, Christian Otten and Waldemar Vollmer, date: 03/20/2018, view: 1271, Q&A: 0
Peptidoglycan encases the bacterial cytoplasmic membrane to protect the cell from lysis due to the turgor. The final steps of peptidoglycan synthesis require a membrane-anchored substrate called lipid II, in which the peptidoglycan subunit is linked to the carrier lipid undecaprenol via a pyrophosphate moiety. Lipid II is the target of ...
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Microbial Mutagenicity Assay: Ames Test Authors:  Urvashi Vijay, Sonal Gupta, Priyanka Mathur, Prashanth Suravajhala and Pradeep Bhatnagar, date: 03/20/2018, view: 5822, Q&A: 2
The Microbial mutagenicity Ames test is a bacterial bioassay accomplished in vitro to evaluate the mutagenicity of various environmental carcinogens and toxins. While Ames test is used to identify the revert mutations which are present in strains, it can also be used to detect the mutagenicity of environmental samples such as drugs, dyes, ...
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Single-step Precision Genome Editing in Yeast Using CRISPR-Cas9 Authors:  Azat Akhmetov, Jon M Laurent, Jimmy Gollihar, Elizabeth C Gardner, Riddhiman K Garge, Andrew D Ellington, Aashiq H Kachroo and Edward M Marcotte, date: 03/20/2018, view: 4339, Q&A: 0
Genome modification in budding yeast has been extremely successful largely due to its highly efficient homology-directed DNA repair machinery. Several methods for modifying the yeast genome have previously been described, many of them involving at least two-steps: insertion of a selectable marker and substitution of that marker for the intended ...
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In situ Hybridization (ISH) in Preparasitic and Parasitic Stages of the Plant-parasitic Nematode Meloidogyne spp. Authors:  Maëlle Jaouannet, Chinh-Nghia Nguyen, Michaël Quentin, Stéphanie Jaubert-Possamai, Marie-Noëlle Rosso and Bruno Favery, date: 03/20/2018, view: 1576, Q&A: 0
The spatio-temporal expression pattern of a gene provides important indications to better understand its biological function. In situ hybridization (ISH) uses a labeled complementary single-stranded RNA or DNA probe to localize gene transcripts in a whole organism, a whole organ or a section of tissue. We adapted the ISH technique to the ...
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CRISPR-mediated Tagging with BirA Allows Proximity Labeling in Toxoplasma gondii Authors:  Shaojun Long, Kevin M. Brown and L. David Sibley, date: 03/20/2018, view: 2516, Q&A: 0
Defining protein interaction networks can provide key insights into how protein complexes govern complex biological problems. Here we define a method for proximity based labeling using permissive biotin ligase to define protein networks in the intracellular parasite Toxoplasma gondii. When combined with CRISPR/Cas9 based tagging, this ...
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Biochemical Analysis of Dimethyl Suberimidate-crosslinked Yeast Nucleosomes Authors:  Yuichi Ichikawa and Paul D. Kaufman, date: 03/20/2018, view: 1536, Q&A: 0
Nucleosomes are the fundamental unit of eukaryotic chromosome packaging, comprised of 147 bp of DNA wrapped around two molecules of each of the core histone proteins H2A, H2B, H3, and H4. Nucleosomes are symmetrical, with one axis of symmetry centered on the homodimeric interaction between the C-termini of the H3 molecules. To explore the ...
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Quantification of Colibactin-associated Genotoxicity in HeLa Cells by In Cell Western (ICW) Using γ-H2AX as a Marker Authors:  Sophie Tronnet and Eric Oswald, date: 03/20/2018, view: 1395, Q&A: 0
The genotoxin colibactin is produced by several species of Enterobacteriaceae. This genotoxin induces DNA damage, cell cycle arrest, senescence and death in eukaryotic cells (Nougayrède et al., 2006; Taieb et al., 2016). Here we describe a method to quantify the genotoxicity of bacteria producing colibactin following a ...
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Alphavirus Purification Using Low-speed Spin Centrifugation Authors:  Vamseedhar Rayaprolu, Jolene Ramsey, Joseph Che-Yen Wang and Suchetana Mukhopadhyay, date: 03/20/2018, view: 1509, Q&A: 0
Chemical and sedimentation procedures are used to purify virus particles. While these approaches are successful for wild-type viruses, they are often not feasible for purifying mutant viruses with assembly defects. We combined two published methods (Atasheva et al., 2013; Moller-Tank et al., 2013), to generate a protocol that ...
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Isolation of Commensal Escherichia coli Strains from Feces of Healthy Laboratory Mice or Rats Authors:  Tingting Ju and Benjamin P. Willing, date: 03/20/2018, view: 1495, Q&A: 0
The colonization abundance of commensal E. coli in the gastrointestinal tract of healthy laboratory mice and rats ranges from 104 to 106 CFU/g feces. Although very well characterized, the family that E. coli belongs to has a very homogeneous 16S rRNA gene sequence, making the identification from 16S rRNA ...
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Molecular Biology

RNA Cap Methyltransferase Activity Assay Authors:  Jackson B. Trotman and Daniel R. Schoenberg, date: 03/20/2018, view: 1285, Q&A: 0
Methyltransferases that methylate the guanine-N7 position of the mRNA 5’ cap structure are ubiquitous among eukaryotes and commonly encoded by viruses. Here we provide a detailed protocol for the biochemical analysis of RNA cap methyltransferase activity of biological samples. This assay involves incubation of cap-methyltransferase-containing ...
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Design of Hybrid RNA Polymerase III Promoters for Efficient CRISPR-Cas9 Function Authors:  Joshua Misa, Cory Schwartz and Ian Wheeldon, date: 03/20/2018, view: 1662, Q&A: 0
The discovery of the CRISPR-Cas9 system from Streptococcus pyogenes has allowed the development of genome engineering tools in a variety of organisms. A frequent limitation in CRISPR-Cas9 function is adequate expression levels of sgRNA. This protocol provides a strategy to construct hybrid RNA polymerase III (Pol III) promoters that ...
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Neuroscience

Organotypic Explants of the Embryonic Rodent Hippocampus: An Accessible System for Transgenesis Authors:  Archana Iyer , Lakshmi Subramanian and Shubha Tole, date: 03/20/2018, view: 1392, Q&A: 0
This protocol describes the technique of ex-vivo electroporation to target embryonic hippocampal progenitors in an organotypic slice preparation. This technique allows gene perturbation for examining developmental processes in the embryonic hippocampus while retaining the environment and connectivity of the cells. Gene perturbation can ...
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Spared Nerve Injury Model of Neuropathic Pain in Mice Authors:  Joseph Cichon, Linlin Sun and Guang Yang, date: 03/20/2018, view: 2358, Q&A: 1
Experimental models of peripheral nerve injury have been developed to study mechanisms of neuropathic pain in living animals. The spared nerve injury (SNI) model in rodents is a partial denervation model, in which the common peroneal and tibial nerves are injured, producing consistent and reproducible tactile hypersensitivity in the skin territory ...
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Stem Cell

Measurement of Mesenchymal Stem Cells Attachment to Endothelial Cells Authors:  Shan Wang, Chris D. Madsen and Yaojiong Wu, date: 03/20/2018, view: 1262, Q&A: 1
Mesenchymal stem cells (MSCs) have shown profound therapeutic potential in tissue repair and regeneration. However, recent studies indicate that MSCs are largely entrapped in lungs after intravenous delivery and die shortly. The underlying mechanisms have been poorly understood. We have provided evidence to show that excess expression and ...
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Systems Biology

Reduced Representation Bisulfite Sequencing in Maize Authors:  Fei-Man Hsu, Chung-Ju Rachel Wang and Pao-Yang Chen, date: 03/20/2018, view: 1672, Q&A: 0
DNA methylation is an epigenetic modification that regulates plant development (Law and Jacobsen, 2010). Whole genome bisulfite sequencing (WGBS) is a state-of-the-art method for profiling genome-wide methylation patterns with single-base resolution (Cokus et al., 2008). However, for an organism with a large genome, e.g., the 2.1 ...
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Quantifying Symmetrically Methylated H4R3 on the Kaposi’s Sarcoma-associated Herpesvirus (KSHV) Genome by ChIP-Seq Authors:  Roxanne C. Strahan, Kayla S. Hiura and Subhash C. Verma, date: 03/20/2018, view: 1566, Q&A: 0
Post-translational modifications to histone tails contribute to the three-dimensional structure of chromatin and play an important role in determining the relative expression of nearby genes. One such modification is symmetric di-methylation of arginine residues, which may exhibit different effects on gene expression including blocking the binding ...
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