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Past Issues in 2015

Volume: 5 Issue: 4

Biochemistry

In vitro Chitin Binding Assay Authors: Geneviève Ball, Frédéric Cadoret and Romé Voulhoux, date: 02/20/2015, view: 10065, Q&A: 0

Chitin is polymer of N-acetylglucosamine (GlcNAc) found in the exoskeleton of arthropods and the fungal cell wall. GlcNAc is also implicated in bacterial development, adherence, and signal transduction but can also be used as a carbon source. In vitro chitin binding assay is performed to determine the affinity of a purified protein to the ...

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Determination of Protein-DNA (ZMYND11-DNA) Interaction by a Label-Free Biolayer Interferometry Assay Authors: Yuan-Yuan Li, Hong Wen, Xiao-Bing Shi and Hai-Tao Li, date: 02/20/2015, view: 11056, Q&A: 0

This protocol describes a robust technique for the measurement of ZMYND11-DNA interaction by a label-free Biolayer Interferometry (BLI). ZMYND11 is a novel histone reader protein that specifically recognizes H3.3K36me3 via its tandem Bromodomain, zinc-finger and PWWP domain (BP). ZMYND11 links the histone-variant-mediated transcription elongation ...

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Immunology

Isolation of Lung Infiltrating Cell in Mice Authors: Eduardo P. Amaral, Elena B. Lassunskaia and Maria Regina D’Império-Lima, date: 02/20/2015, view: 16410, Q&A: 0

Inflammatory lung diseases induce strong leukocyte recruitment into the organ, culminating in pneumonia area formation. Here, we describe the protocol for isolation of lung infiltrating cells. Using this assay, we analyzed the lung cell phenotyping by flow cytometry and spontaneous cytokine production by cultivating lung cells ex vivo ...

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Macrophage Phagocytosis Assay of Staphylococcus aureus by Flow Cytometry Authors: Qin Yan, Sun Hee Ahn and Vance G. Fowler Jr, date: 02/20/2015, view: 12543, Q&A: 0

This protocol describes a straightforward technique to evaluate the phagocytotic capacity of murine macrophages for Staphylococcus aureus (S. aureus). By staining S. aureus with Hexidium Iodide and staining murine bone marrow-derived macrophages (BMDMs) with FITC, the macrophage bacterial up-taking ability can be rapidly ...

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Neuroscience

Preparation of Synaptosomes from the Motor Cortex of Motor Skill Trained Mice Authors: Anand Suresh and Anna Dunaevsky, date: 02/20/2015, view: 12537, Q&A: 0

Learning and memory are thought to occur due to changes in synaptic strength. Strengthening of synapses due to Long Term Potentiation mechanisms are mediated by increases in synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) levels. Here we describe a protocol to isolate and quantify synaptic AMPAR subunit GluA1 levels ...

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Plant Science

Protein Degradation Assays in Arabidopsis Protoplasts Authors: Filip Mituła, Anna Kasprowicz-Maluśki, Michał Michalak, Małgorzata Marczak, Konrad Kuczyński and Agnieszka Ludwików, date: 02/20/2015, view: 12520, Q&A: 2

Plant transformation and exogenous protein expression is essential for molecular biology and biotechnology. Current approaches of stable plant transformation might be problematic and very time-consuming. Because of this, transient expression in protoplasts has become valuable alternative, being less cost and time-effective at the same time. ...

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Phenol-based Total Protein Extraction from Lily Plant Tissues Authors: Yun-Chu Chen, Yu-Chun Hsiao and Co-Shine Wang, date: 02/20/2015, view: 9411, Q&A: 0

The phenol-based total protein extraction method is unique in that water-soluble components such as polyphenolic compounds and nucleic acids can be easily removed. Thus, total protein is free from contaminants and allows for high quality two–dimensional gel electrophoresis. The phenol-based extraction of total protein was used in various lily ...

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Separation of Microspores from Anthers of Lilium longiflorum (Lily) and Subsequent RNA Extraction Authors: Ming-Che Liu, Yun-Chu Chen and Co-Shine Wang, date: 02/20/2015, view: 8534, Q&A: 0

This protocol has been designed in order to facilitate the isolation and extraction of total RNA from microspores collected from lily anther sacs. This protocol allows the extraction of high amounts of high quality RNA, as observed in agarose gels.

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Transmission Electron Microscopy for Tobacco Chloroplast Ultrastructure Authors: Lina Yin, Shiwen Wang, Norihiro Shimomura and Kiyoshi Tanaka, date: 02/20/2015, view: 8528, Q&A: 0

The chloroplast is the site of photosynthesis that enabled and sustains aerobic life on Earth. Chloroplasts are relatively large organelles with a diameter of ~5 μm and width of ~2.5 μm, and so can be readily analysed by electron microscopy. Each chloroplast is enclosed by two envelope membranes, which encompass an aqueous matrix, the stroma and ...

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Isolation of Chloroplast Inner and Outer Envelope Membranes Authors: Shiwen Wang, Lina Yin, Jun’ichi Mano and Kiyoshi Tanaka, date: 02/20/2015, view: 11595, Q&A: 0

The chloroplast is an important organelle found in plant cells that conduct photosynthesis. It is enclosed by a pair of closely spaced membranes, the double-membrane envelope, consisting of the inner membrane bounding the matrix or stroma and the outer membrane in contact with the cytoplasm. Like many bio-membranes, the chloroplast envelope plays ...

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Systems Biology

Whole Genome Bisulfite Sequencing and DNA Methylation Analysis from Plant Tissue Authors: Daniela Pignatta, George W. Bell and Mary Gehring, date: 02/20/2015, view: 17102, Q&A: 0

This protocol describes whole genome bisulfite-sequencing library preparation from plant tissue and subsequent data analysis. Allele-specific methylation analysis and genome-wide identification of differentially methylated regions are additional features of the analysis procedure.

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