Volume: 7 Issue: 19 —BIO-PROTOCOL

Biochemistry

Protein Expression and Purification of the Hsp90-Cdc37-Cdk4 Kinase Complex from Saccharomyces cerevisiae Authors: Kliment A. Verba and David A. Agard, date: 10/05/2017, view: 6529, Q&A: 0

Interactions between Hsp90, its co-chaperone Cdc37 and kinases have been biochemically studied for over three decades and have been shown to be functionally important in organisms from yeast to humans. However, formation of a stable complex for structural studies has been elusive. In this protocol we describe expression and purification of ...

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Cell Biology

Scanning Electron Microscopy of Motile Male Gametes of Land Plants Authors: Karen Sue Renzaglia, Renee A. Lopez and Steven J. Schmitt, date: 10/05/2017, view: 6100, Q&A: 0

The only motile cells produced in land plants are male gametes (spermatozoids), which are reduced to non-flagellated cells in flowering plants and most gymnosperms. Although a coiled architecture is universal, the complexity of land plant flagellated cells varies from biflagellated in bryophytes to thousands of flagella per gametes in the seed ...

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DQ-Red BSA Trafficking Assay in Cultured Cells to Assess Cargo Delivery to Lysosomes Authors: Rituraj Marwaha and Mahak Sharma, date: 10/05/2017, view: 12574, Q&A: 0

Lysosomes are the terminal end of the endocytic pathway having acidic environment required for active hydrolases that degrade the cargo delivered to these compartments. This process of cargo delivery and degradation by endo-lysosomes is a tightly regulated process and important for maintaining cellular homeostasis. Cargos like EGF (Epidermal ...

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Microbiology

A Flow-assay for Farnesol Removal from Adherent Candida albicans Cultures Authors: Melanie Polke and Ilse D. Jacobsen, date: 10/05/2017, view: 5875, Q&A: 0

Here, we describe a protocol for a continuous flow system for C. albicans cultures growing adherent to a plastic surface. The protocol was adapted from a previous method established to simulate blood flow on endothelial cells (Wilson and Hube, 2010). The adapted protocol was used by us for the removal of molecules in C. albicans ...

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Quantification of Densities of Bacterial Endosymbionts of Insects by Real-time PCR Author: Daisuke Kageyama, date: 10/05/2017, view: 7693, Q&A: 0

Increased attention has been paid to the endosymbiotic bacteria of insects. Because most insect endosymbionts are uncultivable, quantitative PCR (qPCR) is a practical and convenient method to quantify endosymbiont titers. Here we report a protocol for real-time qPCR based on SYBR Green I fluorescence as well as some tips to prevent possible ...

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Molecular Biology

Generation of Caenorhabditis elegans Transgenic Animals by DNA Microinjection Authors: Matthias Rieckher and Nektarios Tavernarakis, date: 10/05/2017, view: 10036, Q&A: 0

Microinjection is the most frequently used tool for genetic transformation of the nematode Caenorhabditis elegans, facilitating the transgenic expression of genes, genome editing by the clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 system, or transcription of dsRNA for RNA intereference (RNAi). Exogenous DNA is ...

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Accurate, Streamlined Analysis of mRNA Translation by Sucrose Gradient Fractionation Authors: Soufiane Aboulhouda, Rachael Di Santo, Gabriel Therizols and David Weinberg, date: 10/05/2017, view: 8303, Q&A: 0

The efficiency with which proteins are produced from mRNA molecules can vary widely across transcripts, cell types, and cellular states. Methods that accurately assay the translational efficiency of mRNAs are critical to gaining a mechanistic understanding of post-transcriptional gene regulation. One way to measure translational efficiency is to ...

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Protocol for Construction of a Tunable CRISPR Interference (tCRISPRi) Strain for Escherichia coli Authors: Xin-tian Li, Cindy Sou and Suckjoon Jun, date: 10/05/2017, view: 7486, Q&A: 0

We present a protocol for construction of tunable CRISPR interference (tCRISPRi) strains for Escherichia coli. The tCRISPRi system alleviates most of the known problems of plasmid-based expression methods, and can be immediately used to construct libraries of sgRNAs that can complement the Keio collection by targeting both essential and ...

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Neuroscience

Preparation of Teased Nerve Fibers from Rat Sciatic Nerve Authors: Jinkun Wen, Lixia Li, Dandan Tan and Jiasong Guo, date: 10/05/2017, view: 8760, Q&A: 0

Compared to tissue sectioning techniques, the technique of teasing single nerve fibers provides a better way to understand the structures of myelin sheaths and axons of the peripheral myelinated nerves. This protocol describes a method for preparation of teased single nerve fibers from rat sciatic nerve. In this protocol, fixed nerves are teased ...

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Plant Science

Transmission Electron Microscopy of Centrioles, Basal Bodies and Flagella in Motile Male Gametes of Land Plants Authors: Karen Sue Renzaglia, Renee A. Lopez, Jason S. Henry, Nicholas D. Flowers and Kevin C. Vaughn, date: 10/05/2017, view: 6517, Q&A: 0

Motile male gametes (spermatozoids) of land plants are coiled and contain a modified and precisely organized complement of organelles that includes a locomotory apparatus with two to thousands of flagella. Each flagellum is generated from a basal body that originates de novo as a centriole in spermatogenous cell lineages. Much of what is ...

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Ensifer-mediated Arabidopsis thaliana Root Transformation (E-ART): A Protocol to Analyse the Factors that Support Ensifer-mediated Transformation (EMT) of Plant Cells Authors: Dheeraj Singh Rathore, Fiona M. Doohan and Ewen Mullins, date: 10/05/2017, view: 8127, Q&A: 0

Ensifer adhaerens OV14, a soil borne alpha-proteobacteria of the Rhizobiaceae family, fortifies the novel plant transformation technology platform termed ‘Ensifer-mediated transformation’ (EMT). EMT can stably transform both monocot and dicot species, and the host range of EMT is continuously expanding across a diverse range of ...

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Large-scale Maize Seedling Infection with Exserohilum turcicum in the Greenhouse Authors: Ping Yang, Gerhard Herren, Simon G. Krattinger and Beat Keller, date: 10/05/2017, view: 7541, Q&A: 0

Northern corn leaf blight (NCLB) is a serious foliar disease of maize (Zea mays) worldwide and breeding for resistance is of primary importance for maize crop protection. Phenotyping for NCLB resistance is well established in the field, but such experiments depend on suitable environmental conditions and are seasonal. Here we describe a ...

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Xanthomonas oryzae pv. oryzae Inoculation and Growth Rate on Rice by Leaf Clipping Method Authors: Yinggen Ke, Shugang Hui and Meng Yuan, date: 10/05/2017, view: 12829, Q&A: 0

Bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious bacterial diseases and a major impediment to the increase of rice yield. Appropriate methods for inoculation of Xoo and disease scoring are necessary to investigate the nature of the disease and the mechanism of plant ...

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Analysis of Xyloglucan Composition in Arabidopsis Leaves Authors: Javier Sampedro, Cristina Gianzo, Esteban Guitián, Gloria Revilla and Ignacio Zarra, date: 10/05/2017, view: 4456, Q&A: 0

Xyloglucan is one of the main components of the primary cell wall in most species of plants. This protocol describes a method to analyze the composition of the enzyme-accessible and enzyme-inaccessible fractions of xyloglucan in the model species Arabidopsis thaliana. It is based on digestion with an endoglucanase that attacks ...

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Scanning Electron Microscope (SEM) Imaging to Determine Inflorescence Initiation and Development in Olive Authors: Amnon Haberman, Einat Zelinger and Alon Samach, date: 10/05/2017, view: 6604, Q&A: 0

Here we present a protocol that describes how to image the structure of the olive axillary bud meristem with a scanning electron microscope (SEM) in order to characterize its identity and developmental stage. Briefly, the specimen is fixed with glutaraldehyde, saturated with ethanol, dried in a critical point dryer (CPD) system, dissected, coated ...

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Past Issues in 2017

Volume: 7 Issue: 19

Biochemistry

Cell Biology

Microbiology

Molecular Biology

Neuroscience

Plant Science