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Cancer Biology

Measurement of Endogenous MALT1 Activity Authors:  Daniel Nagel and Daniel Krappmann, date: 07/20/2013, view: 6154, Q&A: 0
MALT1(Mucosa associated lymphoid tissue protein 1) is an important adapter protein for the NF-kB driven lymphocyte activation and the development and survival of distinct B-cell lymphoma entities. In addition MALT1 is a cysteine protease that structurally resembles caspases while having a different substrate preference and mechanism of ...
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Estradiol Receptor (ER) Chromatin Immunoprecipitation in MCF-7 Cells Authors:  Pia Giovannelli, Gabriella Castoria and Antimo Migliaccio, date: 07/20/2013, view: 5027, Q&A: 0
Steroid hormone receptors, for example estradiol receptor, act like transcription factors. In the cell, steroids bind to a specific receptor. Upon ligand binding, many steroid receptors dimerize and enter nuclei where they bind specific DNA sequences called Hormone Responsive Elements (HRE) and regulate gene transcription. ER is able to bind DNA ...
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Immunology

Endosomal pH Measurement in Bone Marrow Derived Dendritic Cells Authors:  Sophia Maschalidi and Bénédicte Manoury, date: 07/20/2013, view: 4568, Q&A: 0
Endosomes embraces different set of compartments such as early endosomes, intermediate endosomes and late endosomes or lysosomes. They become acidic as they mature. This acidification is generated by the vacuolar membrane proton pump V-ATPase that is recruited in late endosomes. This protocol described the measurement of endosomal pH using dextran ...
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Isolation of Phagosomes from Dendritic Cells by Using Magnetic Beads Author:  Bénédicte Manoury, date: 07/20/2013, view: 4496, Q&A: 0
Phagosomes are intracellular organelles in dendritic cells in which pathogens such as viruses, bacteria and parasites are internalised to be proteolysed and killed. Phagosomes are formed by fusion with the plasma membrane, some area of the endoplasmic reticulum as well as the lysosome. This protocol described the purification of phagosomal ...
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Microbiology

Preparation of Candida albicans Biofilms for Transmission Electron Microscopy Authors:  Heather T. Taff and David R. Andes, date: 07/20/2013, view: 5870, Q&A: 0
Transmission Electron Microscopy is a form of microscopy that allows for imaging of distinct portions of an individual cell. For Candida albicans biofilms, it is often used to visualize the cell walls of fixed samples of yeast and hyphae. This protocol describes how to grow, harvest, and fix Candida albicans biofilms in ...
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Preparation of Candida albicans Biofilms Using an in vivo Rat Central Venous Catheter Model Authors:  Heather T. Taff, Karen Marchillo and David R. Andes, date: 07/20/2013, view: 4241, Q&A: 0
In vivo biofilms grown on medical devices are necessary to understand the interactions of the fungal biofilm and the host environment in which it is most commonly found. This protocol describes a way to grow Candida albicans biofilms on the interior lumen of central venous catheters surgically implanted into rats, which mimics ...
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High-throughput β-galactosidase and β-glucuronidase Assays Using Fluorogenic Substrates Author:  Joshua P. Ramsay, date: 07/20/2013, view: 7762, Q&A: 0
β-galactosidase and β-glucuronidase enzymes are commonly used as reporters for gene expression from gene promoter-lacZ or uidA fusions (respectively). The protocol described here is a high-throughput alternative to the commonly used Miller assay (Miller, 1972) that utilises a fluorogenic substrate (Fiksdal et al., 1994) ...
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Extraction and Quantification of Cyclic Di-GMP from Pseudomonas aeruginosa Authors:  Ankita Basu Roy, Olga E. Petrova and Karin Sauer, date: 07/20/2013, view: 6920, Q&A: 1
Cyclic di-GMP (c-di-GMP) has emerged as an important intracellular signaling molecule, controlling the transitions between planktonic (free-living) and sessile lifestyles, biofilm formation, and virulence in a wide variety of microorganisms. The following protocol describes the extraction and quantification of c-di-GMP from Pseudomonas aeruginosa ...
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Analyzing Inhibitory Effects of Reagents on Mycoplasma Gliding and Adhesion Authors:  Taishi Kasai and Makoto Miyata, date: 07/20/2013, view: 6303, Q&A: 0
Dozens of Mycoplasma species bind to solid surfaces and glide in the direction of the membrane protrusion at a pole. In gliding, Mycoplasma legs catch, pull and release sialylated oligosaccharides fixed on a solid surface. The analyses of inhibitory effects of sialylated compounds on gliding of Mycoplasma can determine ...
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HIV-1 Virus-like Particle Budding Assay Authors:  Nathan H Vande Burgt, Luis J Cocka and Paul Bates, date: 07/20/2013, view: 5400, Q&A: 0
Viral replication culminates with the egress of the mature virion from the host cell. This step of the viral life cycle has recently garnered increased attention with the discovery of the cellular restriction factor, Tetherin, which tethers budded virions to the surface of infected cells and inhibits viral spread. The importance of this block in ...
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Molecular Biology

DNase I Footprinting to Identify Protein Binding Sites Authors:  Isabelle Gaugué, Dominique Bréchemier-Baey and Jacqueline Plumbridge, date: 07/20/2013, view: 9894, Q&A: 0
DNase I footprinting is used to precisely localise the position that a DNA binding protein, e.g. a transcription factor, binds to a DNA fragment. A DNA fragment of a few hundred bp is labelled at one end and then incubated with the proteins suspected to bind. After a limited digestion with DNase I, the reaction is quenched, DNA is ...
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Polysome Profiling Analysis Authors:  Masahiro Morita, Tommy Alain, Ivan Topisirovic and Nahum Sonenberg, date: 07/20/2013, view: 20272, Q&A: 2
Polysome profiling is a method that allows monitoring of translation activity of mRNAs in cells and tissues. Once each polysome fractions are collected, the translation activity of each mRNA is analyzed using various molecular biology techniques such as Northern blotting, RT-PCR, microarray or deep-sequencing.
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Plant Science

Analysis of Malondialdehyde, Chlorophyll Proline, Soluble Sugar, and Glutathione Content in Arabidopsis seedling Authors:  Zhijin Zhang and Rongfeng Huang, date: 07/20/2013, view: 15578, Q&A: 0
The protocol has four sub-protocols, which are about the measurement of malondialdehyde, chlorophyll proline, soluble sugar, and glutathione content, respectively, in Arabidopsis seedling by using spectrophotometer. These methods are simple, effective and reproducible, which will help the researchers who are not familiar with these ...
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Determination of Enzyme Kinetic Parameters of UDP-glycosyltransferases Authors:  Jörg M. Augustin and Søren Bak, date: 07/20/2013, view: 5518, Q&A: 0
The determination of enzyme kinetic parameters, such as the Km and kcat values, is an essential part of the characterization of newly discovered enzymes. This protocol describes the determination of enzyme kinetic parameters of the Barbarea vulgaris UDP-glycosyltransferases (UGTs) UGT73C11 and UGT73C13 toward the ...
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Extraction and Reglucosylation of Barbarea vulgaris Sapogenins Authors:  Jörg M. Augustin, Carl Erik Olsen and Søren Bak, date: 07/20/2013, view: 5003, Q&A: 0
Plants produce a vast array of natural compounds. Many of them are not commercially available, and are thus lacking to be tested as substrates for enzymes. This protocol describes the extraction and acidic hydrolysis of metabolites from Barbarea vulgaris with special focus on saponins and their agylcones (sapogenins). It was developed to ...
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Maize Endosperm Protein Extraction and Analysis Authors:  Xinze Chen, Dongsheng Yao and Rentao Song, date: 07/20/2013, view: 5656, Q&A: 0
Alcohol-solubility is the most characteristic feature of the zein proteins, the major storage protein in maize. Using sodium borate buffer system with added reducing agent, total proteins are isolated, and zein proteins are separated from non-zein proteins. The extraction effect is intuitive on a SDS-PAGE isolation system. In addition, a simple ...
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