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Biochemistry

Protein Flotation Assay to Isolate Lipids Rafts from Soft Tissue or Cells Authors:  Catherine Lemaire-Vieille, Jean Gagnon and Jean-Yves Cesbron, date: 08/20/2013, view: 5483, Q&A: 0
The arrangement in eukaryotic cell membranes of liquid-ordered states surrounded by liquid-disordered phases allows for the existence of organized membrane microdomains called rafts. Rafts play a crucial role in signal-transduction events, in lipid transport and in various internalization processes, and for all these reasons need to be purified ...
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PTEN-lipid Binding Assay Authors:  Sridhar Kavela, Swapnil R. Shinde and Subbareddy Maddika, date: 08/20/2013, view: 4801, Q&A: 0
The lipid and protein interactions are an integral and important part of many cellular signaling pathways. The understanding of the selective and specific interaction of the given lipid molecule with the target protein is required for studying cellular signaling. In this assay, different lipids are spotted onto a nitrocellulose membrane to which ...
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Cancer Biology

Natural Killer Cell Transfer Assay Authors:  Lee-Hwa Tai, Christiano Tanese de Souza, Andrew P. Makrigiannis and Rebecca Ann C. Auer, date: 08/20/2013, view: 5644, Q&A: 0
Natural Killer (NK) cells are cytotoxic lymphocytes that constitute a major component of the innate immune system. Immunosurveillance of the host by NK cells for malignant and virally-infected cells results in direct cytotoxicity and the production of cytokines to enhance the immune response. This protocol will describe the adoptive transfer of ...
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Quantitative Methylation Specific PCR (qMSP) Authors:  Triantafillos Liloglou and Georgios Nikolaidis, date: 08/20/2013, view: 14513, Q&A: 0
Detection of low copies of methylated DNA targets in clinical specimens is challenging. The quantitative Methylation-Specific PCR (qMSP) assays were designed to specifically amplify bisulphite-converted methylated DNA target sequences in the presence of an excess of unmethylated counterpart sequences. These qMSP assays are real-time PCR assays ...
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Cell Biology

Cell Surface Protein Biotinylation and Analysis Authors:  Anna Tarradas, Elisabet Selga, Helena Riuró, Fabiana S. Scornik, Ramon Brugada and Marcel Vergés, date: 08/20/2013, view: 11851, Q&A: 0
A great way to specifically isolate and quantify proteins in the cell surface membrane is to take advantage of the biotinylation technique. It consists of labeling cell surface proteins with a biotin reagent before lysing the cells, and isolating these tagged proteins by NeutrAvidin pull-down. Then, the samples are subjected to SDS-PAGE ...
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Sodium Current Measurements in HEK293 Cells Authors:  Helena Riuró, Anna Tarradas, Elisabet Selga, Ramon Brugada, Fabiana Scornik and Guillermo Pérez, date: 08/20/2013, view: 7000, Q&A: 0
This protocol is used to measure sodium currents from heterologously transfected cells lines, such as HEK293 cells. Standard whole cell patch clamp technique is used to assess ion channel function. This protocol has been used to study cardiac type Nav1.5 sodium channel, and in particular to compare wild-type and mutant channels related ...
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Fluorescence in situ Hybridization to the Polytene Chromosomes of Anopheles Mosquitoes Authors:  Ai Xia, Ashley Peery, Maryam Kamali, Jiangtao Liang, Maria V. Sharakhova and Igor V. Sharakhov, date: 08/20/2013, view: 6665, Q&A: 0
Fluorescence in situ hybridization (FISH) is a method that uses a fluorescently labeled DNA probe for mapping the position of a genetic element on chromosomes. A DNA probe is prepared by incorporating Cy-3 or Cy-5 labeled nucleotides into DNA by nick-translation or a random primed labeling method. This protocol was used to map genes ...
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Immunology

Bronchoalveolar Lavage and Lung Tissue Digestion Authors:  Hongwei Han and Steven F. Ziegler, date: 08/20/2013, view: 19509, Q&A: 0
Bronchoalveolar lavage (BAL) is a simple but valuable and typically performed technique commonly used for studying the pathogenesis of lung diseases such as asthma and COPD. Cell counts can be combined with new methods for examining inflammatory responses, such as ELISA, Flow cytometric analysis, immunohistochemistry, quantitative polymerase chain ...
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Ex vivo Natural Killer Cell Cytotoxicity Assay Authors:  Lee-Hwa Tai, Christiano Tanese de Souza, Andrew P. Makrigiannis and Rebecca Ann C. Auer, date: 08/20/2013, view: 8543, Q&A: 0
Natural Killer (NK) cells are cytotoxic lymphocytes that constitute a major component of the innate immune system. Immunosurveillance of the host by NK cells for malignant and virally-infected cells results in direct cytotoxicity and the production of cytokines to enhance the immune response. This protocol will describe the “gold standard” ...
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Microbiology

Zymogram Assay for the Detection of Peptidoglycan Hydrolases in Streptococcus mutans Authors:  Delphine Dufour and Céline M. Lévesque, date: 08/20/2013, view: 7050, Q&A: 1
Peptidoglycan hydrolases or autolysins are enzymes capable of cleaving covalent bonds in bacterial peptidoglycan cell wall layer. They can participate in the cell division process, in the release of turnover products from peptidoglycan during cell growth, and in cell autolysis induced under particular conditions. The protocol for zymogram ...
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Drug Sensitivity Assay of Xanthomonas citri subsp. citri Using REMA Plate Method Authors:  Isabel C. Silva and Henrique Ferreira, date: 08/20/2013, view: 5140, Q&A: 0
Resazurin Microtiter Assay (REMA) is a simple, rapid, reliable, sensitive, safe and cost-effective measurement of cell viability. Resazurin detects cell viability by converting from a nonfluorescent dye to the highly red fluorescent dye resorufin in response to chemical reduction of growth medium resulting from cell growth (Palomino et al. ...
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Metabolic Labeling of Yeast Sphingolipids with Radioactive D-erythro-[4,5-3H]dihydrosphingosine Authors:  Takefumi Karashima, Kentaro Kajiwara and Kouichi Funato, date: 08/20/2013, view: 5076, Q&A: 0
Yeast sphingolipids can be metabolically labeled with exogenously added radioactive precursors. Here we describe a method to label ceramides and complex sphingolipids of Saccharomyces cerevisiae with a radioactive ceramide precursor, D-erythro-[4, 5-3H]dihydrosphingosine. This protocol is used to study the biosynthesis, ...
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Isolation of Radiolabeled Poliovirus Particles from H1 HeLa Cells Authors:  Alexsia Richards and William Jackson, date: 08/20/2013, view: 4145, Q&A: 0
The following protocol describes the isolation of radioactive viral and subviral particles from infected cells. This protocol has been written for isolation of poliovirus particles from H1 HeLa cells. Infection protocol and timing of [35S] methionine labeling and particle collection should be tailored to the virus of interest. ...
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Bioassay to Screen Pathogenesis-associated Genes in Alternaria brassicicola Author:  Yangrae Cho, date: 08/20/2013, view: 4524, Q&A: 0
Alternaria brassicicola is a necrotrophic fungus that causes black spot disease of most plants in the Brassicaceae, including cultivated Brassica species and weedy Arabidopsis species. Since the concept of transformation constructs of linear minimal elements was developed (Cho et al., 2006), we have ...
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Molecular Biology

Ultra-low Background DNA Cloning System Authors:  Yukio Nagano and Kenta Goto, date: 08/20/2013, view: 9777, Q&A: 0
We have developed a method to clone DNA fragments into the E. coli plasmid vectors with almost 100% efficiency (Goto and Nagano, 2013). This method is based on highly efficient yeast-based in vivo cloning, and the subsequent cloning of the constructed plasmids into E. coli. Our method is useful for various applications: ...
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Neuroscience

Primary Culture of SVZ-derived Progenitors Grown as Neurospheres Authors:  Julien Vernerey, Karine Magalon and Pascale Durbec, date: 08/20/2013, view: 5697, Q&A: 0
SVZ-derived progenitors grown as neurospheres is a well-known model to study neural stem cell and progenitor functions such as proliferation, differentiation/self-renewal, and migration (Durbec and Rougon, 2001). This protocol is for preparing a culture of SVZ-derived progenitors from 8 early postnatal mouse brains (P0 to P3). One week after cell ...
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Retrograde and Anterograde Tracing of Neural Projections Authors:  Takehiro Kudo and Masahiko Watanabe, date: 08/20/2013, view: 8722, Q&A: 0
Neurons consist of four elements, the soma, dendrite, axon and terminal. They work in concert as the input (soma and dendrite) and output (axon and terminal) parts of neuronal transmission. To function and maintain neuronal activity and metabolisms, proteins and organelles should be transported from soma to terminal via anterograde axonal ...
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Plant Science

Reverse Zymmogram Analysis for the Detection of Protease Inhibitor Activity Authors:  Lilia Bernal, Florencia García-Campusano, Edgar Nájera and Felipe Cruz-García, date: 08/20/2013, view: 5927, Q&A: 0
This protocol describes a gel-based procedure to detect protease inhibitor activity. In this method gelatin is used as a substrate for proteolysis and is copolymerized within the polyacrylamide matrix. Protein extracts are fractioned by SDS-PAGE and then the gel is treated with the protease of interest, which degrades gelatin, except in the areas ...
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Maize Embryo Transient Transformation by Particle Bombardment Author:  Matilde Jose-Estanyol, date: 08/20/2013, view: 4512, Q&A: 0
Particle bombardment has been shown to be a useful method to study gene promoter regulatory elements by transient transformation of maize embryos with different constructions of gene promoters fused to a gene reporter. DNA to transfer is coated to high density gold microparticles and introduced into cells when accelerated by a helium pulse. This ...
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Measuring Germination Percentage in Wheat Authors:  Harish Manmathan and Nora L.V. Lapitan, date: 08/20/2013, view: 9613, Q&A: 0
Knowledge of the viability of seeds is a prerequisite for establishing the seeding rate in crop production and for germination-related trait evaluation in crop plants. This method explains a simple procedure to establish germination percentage in wheat seeds.
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Rapid Induction of Water Stress in Wheat Authors:  Harish Manmathan and Nora L.V. Lapitan, date: 08/20/2013, view: 5504, Q&A: 0
Traditional water stress evaluation studies in wheat are time consuming and can take up to several months to finish. A rapid phenotypic screening for water stress is important for accommodating time-bound water stress studies such as transient gene silencing studies in wheat. This method explains a procedure to induce water stress in young wheat ...
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Determination of Nitrate Uptake and Accumulation Using 15N in Rice Seedlings Authors:  Zhong Tang and Guohua Xu, date: 08/20/2013, view: 3917, Q&A: 0
Nitrogen-15 is a rare stable isotope of nitrogen. This isotope is often used in agricultural research. For example, Nitrogen-15 is used to trace mineral nitrogen compounds and translocate the nitrogen molecule in plants. This protocol is used to determine nitrate uptake and accumulation in rice seedlings by using Nitrogen-15.
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Measurement of Net NO3- Flux in Rice Plants with the SIET System Authors:  Zhong Tang and Guohua Xu, date: 08/20/2013, view: 3941, Q&A: 0
SIET (scanning ion-electrode technique) is a new technique to study the flow rate of the ions and molecules in real time in living biomaterials by using microelectrodes and microsensor. This technique allows non-invasive, simultaneous measurement of fluxes of specific ions at the surface of an intact plant. It has high temporal and spatial ...
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