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Past Issues in Bio-protocol

Volume: 5 Issue: 16August 20, 2015

  • All fields (15)
  • Biochemistry (1)
  • Cancer Biology (2)
  • Cell Biology (1)
  • Immunology (2)
  • Microbiology (2)
  • Plant Science (5)
  • Stem Cell (1)
  • Systems Biology (1)

Biochemistry

Sample Preparation of Telomerase Subunits for Crystallization Authors:  Jing Huang, Christopher J. Bley, Dustin P. Rand, Julian J.-L. Chen and Ming Lei, date: 08/20/2015, view: 2058, Q&A: 0
Telomerase is a large ribonucleoprotein complex that replicates the linear chromosome ends in most eukaryotes. Large-scale preparation of the telomerase core components in vitro has long been a big challenge in this field, hindering the understanding of the catalytic mechanism of telomerase, as well as slowing down the development of ...
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Cancer Biology

Adoptive Transfer of Myeloid-Derived Suppressor Cells and T Cells in a Prostate Cancer Model Authors:  Libo Yan and Yan Xu, date: 08/20/2015, view: 2333, Q&A: 0
The adoptive transfer of immune cells for cancer, chronic infection, and autoimmunity is an emerging field that has shown promise in recent trials. The transgenic adenocarcinoma mouse prostate (TRAMP) is a classical mouse model of prostate cancer (PCa) and TRAMP cell lines were derived from a TRAMP mouse tumor. TRAMP-C2 is tumorigenic when ...
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Determining Leukocyte Origins Using Parabiosis in the PyMT Breast Tumor Model Authors:  Ruth A. Franklin and Ming O. Li, date: 08/20/2015, view: 3007, Q&A: 0
Tumors develop in a complex microenvironment alongside numerous cell types that impact their survival. Immune cells make up a large proportion of these accessory cells and many are known to promote tumor progression. Macrophages, in particular, are associated with poor patient prognosis and are therefore potential candidates for therapeutic ...
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Cell Biology

Labeling of the Intestinal Lumen of Caenorhabditis elegans by Texas Red-dextran Feeding Authors:  Keiko Saegusa and Ken Sato, date: 08/20/2015, view: 2478, Q&A: 0
In this method, the intestinal lumen of Caenorhabditis elegans (C. elegans) is labeled with a fluorescent fluid-phase marker, Texas Red-dextran. Since dextran conjugates are membrane impermeable, animals fed with it show a red fluorescent signal in the lumen of the intestine. Texas Red-dextran in the lumen is not efficiently ...
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Immunology

Adoptive Transfer of Memory B Cells Authors:  Griselda Zuccarino-Catania and Mark Shlomchik, date: 08/20/2015, view: 3009, Q&A: 0
The adoptive transfer of antigen-specific B cells into mice that cannot recognize that specific antigen has two main advantages. The first is determining exactly when the B cells were transferred and exposed to antigen. The second is that all B cells that can bind that antigen are the ones that were transferred; no new antigen-specific B cells ...
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Separation of Intracellular Vesicles for Immunoassays Authors:  Toshihiko Kobayashi, Tsubasa Tanaka and Noriko Toyama-Sorimachi, date: 08/20/2015, view: 2785, Q&A: 0
The endosome/lysosome systems play important roles in immune cell functions as signaling platforms. Immune cells utilize these endosome/lysosome for signal transduction or intercellular communication to elicit the proper immune responses, regulating the localization or the association of the signaling complexes. Here we introduce the procedures to ...
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Microbiology

RNase H Polymerase-independent Cleavage Assay for Evaluation of RNase H Activity of Reverse Transcriptase Enzymes Authors:  Angela Corona and Enzo Tramontano, date: 08/20/2015, view: 2636, Q&A: 0
The ribonuclease H (RNase H) polymerase-independent cleavage assay allows detection and quantification of RNase H activity of reverse transcriptase (RT) enzymes with a hybrid substrate formed by a fluorescein labeled RNA annealed with Dabcyl DNA (Figure 1). Here we describe a protocol that we have adapted for HIV-1 RT expressed from a ...
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Isolation of Rhizosphere Bacterial Communities from Soil Authors:  Laura J. White, Volker S. Brözel and Senthil Subramanian, date: 08/20/2015, view: 6014, Q&A: 0
Rhizosphere bacterial communities have become a major focal point of research in recent years, especially regarding how they affect plants and vice versa (Philippot et al., 2013). Changes in microbial density and diversity within the rhizosphere occur in a spatial temporal manner. The soil zone closest to the plant roots has the most ...
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Plant Science

High-throughput Quantification of Ammonium Content in Arabidopsis Authors:  Izargi Vega-Mas, Asier Sarasketa and Daniel Marino, date: 08/20/2015, view: 2314, Q&A: 0
This protocol is a simple colorimetric assay for internal ammonium quantification in aqueous extracts from plant tissues. The method is based on the phenol hypochlorite assay (Berthelot reaction):

NH4+ + hypochlorite + OH- + phenol → indophenol

The oxidation of indophenol caused by phenol ...
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In vitro Colorimetric Method to Measure Plant Glutamate Dehydrogenase Enzyme Activity Authors:  Asier Sarasketa, Izargi Vega-Mas and Daniel Marino, date: 08/20/2015, view: 2877, Q&A: 0
Glutamate dehydrogenase (GDH) is an NAD(H) dependent enzyme that catalyzes, in vitro, the reversible amination of glutamate. Here we describe how to determine spectrophotometrically GDH activity monitoring NADH evolution. This protocol is described here for Arabidopsis thaliana (A. thaliana) although it is also valid for ...
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Glucosinolates Determination in Tissues of Horseradish Plant Authors:  Filomena Lelario, Susanna De Maria, Rosa Agneta, Christian Mӧllers, Sabino Aurelio Bufo and Anna Rita Rivelli, date: 08/20/2015, view: 2060, Q&A: 0
Glucosinolates (GLS) are secondary metabolites mainly found in plants belonging to the Brassicaceae family, including also horseradish (Armoracia rusticana G. Gaertn., B. Mey. & Scherb), a popular spice with a characteristic pungent flavor due to the abundance of GLS. Such compounds exhibit antibacterial, antifungal, and insecticidal activities, ...
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Protocol to Treat Seedlings with Brassinazole and Measure Hypocotyl Length in Arabidopsis thaliana Authors:  Ana Espinosa-Ruiz, Cristina Martínez and Salomé Prat, date: 08/20/2015, view: 3199, Q&A: 0
The plant hormones brassinosteroids (BR) promote hypocotyl elongation of Arabidopsis thaliana (A. thaliana) seedlings both under light and dark (etiolated) conditions. A common assay to determine if a mutant or transgenic line is affected in BR biosynthesis or response is a sensitivity assay to brassinazole (BRZ), an inhibitor of ...
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Quantification of Sodium Accumulation in Arabidopsis thaliana Using Inductively Coupled Plasma Optical Emission Spectrometery (ICP-OES) Authors:  Won-Gyu Choi and Simon Gilroy, date: 08/20/2015, view: 2508, Q&A: 0
Salt stress is a major issue for plants growing in both natural and agricultural settings (Deinlein et al., 2014). For example, irrigation can lead to the build up of salts in the soil as the irrigation water evaporates, leading to salinization, inhibition of plant growth, reduced productivity and eventually to loss of agriculturally ...
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Stem Cell

FACS-based Satellite Cell Isolation From Mouse Hind Limb Muscles Authors:  Anastasia Gromova, Matthew T Tierney and Alessandra Sacco, date: 08/20/2015, view: 3756, Q&A: 0
Fluorescence Activated Cell Sorting (FACS) is a sensitive and accurate method for purifying satellite cells, or muscle stem cells, from adult mouse skeletal muscle (Liu et al., 2013; Sacco et al., 2008; Tierney et al., 2014). Mechanical and enzymatic digestion of hind limb muscles releases mononuclear muscle cells into ...
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Systems Biology

Character-State Reconstruction to Infer Ancestral Protein-Protein Interaction Patterns Authors:  Florian Rümpler, Günter Theißen and Rainer Melzer, date: 08/20/2015, view: 3209, Q&A: 0
Protein-protein interactions are at the core of a plethora of developmental, physiological and biochemical processes. Consequently, insights into the origin and evolutionary dynamics of protein-protein interactions may provide information on the constraints and dynamics of specific biomolecular circuits and their impact on the organismal ...
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