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Past Issues in 2014

Volume: 4 Issue: 18

Biochemistry

Dimethylmethylene Blue Assay (DMMB) Authors: Vivien Jane Coulson- Thomas and Tarsis Ferreira Gesteira, date: 09/20/2014, view: 47055, Q&A: 8

Glycosaminoglycans (GAGs) are long unbranched polysaccharides consisting of repeating disaccharide units composed of a hexosamine (glucosamine or galactosamine) and a hexuronic acid (glucuronic or iduronic acid). Depending on the disaccharide unit the GAGs can be organized into five groups: chondroitin sulfate, dermatan sulfate, heparan sulfate, ...

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Protein-lipid Interaction Analysis by Surface Plasmon Resonance (SPR) Authors: Olga Lucia Baron and David Pauron, date: 09/20/2014, view: 10188, Q&A: 0

Interactions of lipids with proteins are essential events in the framework of biological membranes. Assessment of the affinity and specificity of protein-lipid binding can give useful information to elucidate cell membrane functions. Surface Plasmon Resonance (SPR) is a powerful technology to study macromolecular interactions, allowing direct and ...

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Immunology

Isolation and FACS Analysis on Mononuclear Cells from CNS Tissue Authors: Kingston H.G. Mills, Róisín M. McManus and Lara Dungan, date: 09/20/2014, view: 10465, Q&A: 0

Immune cells, such as microglia are resident in the brain and spinal cord of normal mice and humans. Furthermore, macrophages, dendritic cells, T cells, B cells and NK cells infiltrate the CNS during certain infections or in neurodegenerative/neuroinflammatory diseases, such as experimental autoimmune encephalomyelitis (EAE) (a model for multiple ...

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Generation of Aβ-specific T cell lines and in vivo Transfer Authors: Róisín M. McManus, Marina A. Lynch and Kingston H.G. Mills, date: 09/20/2014, view: 7321, Q&A: 0

Amyloid-β (Aβ)-containing plaques accumulate in the brains of patients with Alzheimer’s disease (AD). Studies in transgenic mice which over-express amyloid precursor protein and presenilin 1 (APP/PS1 mice) have suggested that T cells that infiltrate the brain may influence the development of Aβ plaques and associated cognitive dysfuncation. Active ...

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Microbiology

Activity Assays for Bacteriophage Endolysin PlyPy Authors: Rolf Lood and Vincent A. Fischetti, date: 09/20/2014, view: 8191, Q&A: 0

Bacterial viruses (bacteriophages) escape and kill their host by degrading the bacterial peptidoglycan layer through the mechanism of enzymes called endolysins: peptidoglycan degrading enzymes. The method included here is useful for the initial characterization of any endolysin, regardless of the specific catalytic domain (as long as the activity ...

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In vitro Transcription (IVT) and tRNA Binding Assay Author: Sonja MK Schoenfelder, date: 09/20/2014, view: 11152, Q&A: 0

This protocol describes the coupling of (i) “live” in vitro RNA transcription with (ii) binding by a radiolabeled, pre-formed tRNA followed by native gel electrophoresis and phosphorimager scan to visualize the complex. The necessity arose from the stable structure that one RNA forms in the absence of its interaction partner. The T-box ...

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Gentamicin Protection Assay to Determine Bacterial Survival within Macrophages Authors: Sargurunathan Subashchandrabose and Harry L.T. Mobley, date: 09/20/2014, view: 18767, Q&A: 0

Macrophages are key cells involved in orchestrating host defense against infections. Here, we describe the protocol for a bacterial killing assay in macrophages that can be adapted to any bacterial pathogen. Using this assay, we analyzed the survival of wild-type and mutant strains of Escherichia coli (E. coli) within RAW 264.7 ...

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Protocol for Biotin Bioassay-based Cross Feeding Authors: Youjun Feng, Jie Xu, Huimin Zhang, Zeliang Chen and Swaminath Srinivas, date: 09/20/2014, view: 6143, Q&A: 0

Biotin bioassay-based cross-feeding experiments were performed to elucidate the effect on biotin production by bioRbme expression in Agrobacterium tumefaciens (A. tumefaciens) (Feng et al., 2013). The indicator strain used here is the biotin auxotrophic strain of Escherichia coli (E. coli), ...

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Detection of Hog1 Phosphorylation in Candida albicans in Response to an Antifungal Protein Authors: Brigitte ME Hayes and Nicole L van der Weerden, date: 09/20/2014, view: 7071, Q&A: 0

It is becoming increasingly apparent that stress signalling is important for tolerance of fungal species to antifungal chemicals and proteins. The high-osmolarity glycerol (HOG) pathway responds to a number of stressors including osmotic and oxidative stress. This protocol describes a method to detect activation of the Candida albicans ( ...

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Neuroscience

Combined in situ Hybridization/Immunohistochemistry (ISH/IH) on Free-floating Vibratome Tissue Sections Author: Manuel E. Lopez, date: 09/20/2014, view: 11471, Q&A: 0

In situ hybridization and immunostaining are common techniques for localizing gene expression, the mRNA and protein respectively, within tissues. Both techniques can be applied to tissue sections to achieve similar goals, but in some cases, it is necessary to use them together. For example, complement C1q is a secreted protein complex ...

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Plant Science

Extraction of Chloroplast Proteins from Transiently Transformed Nicotiana benthamiana Leaves Author: Joern Klinkenberg, date: 09/20/2014, view: 15081, Q&A: 0

This rapid protocol allows the extraction of chloroplast enriched proteins from Nicotiana benthamiana (N. benthamiana) leaves that were transiently transformed to express an epitope tagged protein of interest. Thus, it can serve as a tool to study the chloroplastidic localization of the protein of interest when it is combined ...

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Thin Sections of Technovit 7100 Resin of Rice Endosperm and Staining Author: Ryo Matsushima, date: 09/20/2014, view: 11228, Q&A: 0

Starch is a biologically and commercially important carbohydrate that is accumulated in plant storage organs, such as seed endosperm. Starch is water-insoluble and forms transparent grains, referred to as starch grains (SGs). SGs are easily stained by iodine solution and can be observed under a normal microscope. Technovit 7100 resin is suitable ...

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Perls Staining for Histochemical Detection of Iron in Plant Samples Authors: Tzvetina Brumbarova and Rumen Ivanov, date: 09/20/2014, view: 18270, Q&A: 2

Visualization of iron (Fe) localization in plants has greatly enhanced our understanding of plant Fe homeostasis. One of the relatively simple and yet powerful techniques is the classical Perls blue stain (Perls, 1867). The technique is based on the conversion of ferrocyanide to insoluble crystals of Prussian blue in the presence of Fe³⁺ ...

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