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Volume:3  Issue:14

July 20, 2013

Cancer Biology

Measurement of Endogenous MALT1 Activity

Authors: Daniel Nagel
Daniel NagelAffiliation: Cellular Signal Integration, Helmholtz Zentrum München, Munich, Germany
Bio-protocol author page: a691
 and Daniel Krappmann
Daniel KrappmannAffiliation: Cellular Signal Integration, Helmholtz Zentrum München, Munich, Germany
For correspondence: daniel.krappmann@helmholtz-muenchen.de
Bio-protocol author page: a460
date: 7/20/2013, 4365 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.821 

[Abstract] 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 ...

Article identifier: bio-protocol.org/e821

Estradiol Receptor (ER) Chromatin Immunoprecipitation in MCF-7 Cells

Authors: Pia Giovannelli
Pia GiovannelliAffiliation: Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Naples, Italy
For correspondence: pia.giovannelli@unina2.it
Bio-protocol author page: a696
Gabriella Castoria
Gabriella CastoriaAffiliation: Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Naples, Italy
Bio-protocol author page: a697
 and Antimo Migliaccio
Antimo MigliaccioAffiliation: Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Naples, Italy
Bio-protocol author page: a392
date: 7/20/2013, 3515 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.831 

[Abstract] 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 ...

Article identifier: bio-protocol.org/e831

Immunology

Endosomal pH Measurement in Bone Marrow Derived Dendritic Cells

Authors: Sophia Maschalidi
Sophia MaschalidiAffiliation 1: INSERM, Unité 1013, Paris, France
Affiliation 2: Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Paris, France
Bio-protocol author page: a685
 and Bénédicte Manoury
Bénédicte ManouryAffiliation 1: INSERM, Unité 1013, Paris, France
Affiliation 2: Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Paris, France
For correspondence: benedicte.manoury@inserm.fr
Bio-protocol author page: a346
date: 7/20/2013, 3059 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.819 

[Abstract] 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 ...

Article identifier: bio-protocol.org/e819

Isolation of Phagosomes from Dendritic Cells by Using Magnetic Beads

Author: Bénédicte Manoury
Bénédicte ManouryAffiliation 1: INSERM, Unité 1013, Paris, France
Affiliation 2: Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Paris, France
For correspondence: benedicte.manoury@inserm.fr
Bio-protocol author page: a346
date: 7/20/2013, 3041 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.820 

[Abstract] 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 ...

Article identifier: bio-protocol.org/e820

Microbiology

Preparation of Candida albicans Biofilms for Transmission Electron Microscopy

Authors: Heather T. Taff
Heather T. TaffAffiliation: Department of Medicine, University of Wisconsin, Madison, USA
For correspondence: htaff@wisc.edu
Bio-protocol author page: a333
 and David R. Andes
David R. AndesAffiliation: Department of Medicine, University of Wisconsin, Madison, USA
For correspondence: dra@medicine.wisc.edu
Bio-protocol author page: a689
date: 7/20/2013, 4140 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.822 

[Abstract] 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 ...

Article identifier: bio-protocol.org/e822

Preparation of Candida albicans Biofilms Using an in vivo Rat Central Venous Catheter Model

Authors: Heather T. Taff
Heather T. TaffAffiliation: Department of Medicine, University of Wisconsin, Madison, USA
For correspondence: htaff@wisc.edu
Bio-protocol author page: a333
Karen Marchillo
Karen MarchilloAffiliation: Department of Medicine, University of Wisconsin, Madison, USA
Bio-protocol author page: a690
 and David R. Andes
David R. AndesAffiliation: Department of Medicine, University of Wisconsin, Madison, USA
For correspondence: dra@medicine.wisc.edu
Bio-protocol author page: a689
date: 7/20/2013, 2895 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.823 

[Abstract] 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 ...

Article identifier: bio-protocol.org/e823

High-throughput β-galactosidase and β-glucuronidase Assays Using Fluorogenic Substrates

Author: Joshua P. Ramsay
Joshua P. RamsayAffiliation: School of Biomedical Sciences, Curtin University, Perth, Australia
For correspondence: josh.ramsay@curtin.edu.au
Bio-protocol author page: a450
date: 7/20/2013, 5330 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.827 

[Abstract] 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, ...

Article identifier: bio-protocol.org/e827

Extraction and Quantification of Cyclic Di-GMP from Pseudomonas aeruginosa

Authors: Ankita Basu Roy
Ankita Basu RoyAffiliation: Biological Sciences Department, Binghamton University, State University of New York at Binghamton, Binghamton, USA
Bio-protocol author page: a699
Olga E. Petrova
Olga E. PetrovaAffiliation: Biological Sciences Department, Binghamton University, State University of New York at Binghamton, Binghamton, USA
Bio-protocol author page: a700
 and Karin Sauer
Karin SauerAffiliation: Biological Sciences Department, Binghamton University, State University of New York at Binghamton, Binghamton, USA
For correspondence: ksauer@binghamton.edu
Bio-protocol author page: a701
date: 7/20/2013, 4969 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.828 

[Abstract] 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 ...

Article identifier: bio-protocol.org/e828

Analyzing Inhibitory Effects of Reagents on Mycoplasma Gliding and Adhesion

Authors: Taishi Kasai
Taishi KasaiAffiliation: Department of Biology, Graduate School of Science, Osaka City University, Osaka, Japan
Bio-protocol author page: a712
 and Makoto Miyata
Makoto MiyataAffiliation: Department of Biology, Graduate School of Science, Osaka City University, Osaka, Japan
For correspondence: miyata@sci.osaka-cu.ac.jp
Bio-protocol author page: a711
date: 7/20/2013, 4235 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.829 

[Abstract] 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 ...

Article identifier: bio-protocol.org/e829

HIV-1 Virus-like Particle Budding Assay

Authors: Nathan H Vande Burgt
Nathan H Vande BurgtAffiliation: Department of Microbiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, USA
Bio-protocol author page: a693
Luis J Cocka
Luis J Cocka Affiliation: Department of Microbiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, USA
For correspondence: lcocka@mail.med.upenn.edu
Bio-protocol author page: a694
 and Paul Bates
Paul BatesAffiliation: Department of Microbiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, USA
Bio-protocol author page: a344
date: 7/20/2013, 3657 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.830 

[Abstract] 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 ...

Article identifier: bio-protocol.org/e830

Molecular Biology

DNase I Footprinting to Identify Protein Binding Sites

Authors: Isabelle Gaugué
Isabelle GauguéAffiliation: FRE3630-CNRS (ex UPR9073), Institut de Biologie Physico-Chimique, Paris, France
Bio-protocol author page: a686
Dominique Bréchemier-Baey
Dominique Bréchemier-BaeyAffiliation: FRE3630-CNRS (ex UPR9073), Institut de Biologie Physico-Chimique, Paris, France
Bio-protocol author page: a687
 and Jacqueline Plumbridge
Jacqueline PlumbridgeAffiliation: FRE3630-CNRS (ex UPR9073), Institut de Biologie Physico-Chimique, Paris, France
For correspondence: jackie.plumbridge@ibpc.fr
Bio-protocol author page: a688
date: 7/20/2013, 5925 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.824 

[Abstract] 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 ...

Article identifier: bio-protocol.org/e824

Polysome Profiling Analysis

Authors: Masahiro Morita
Masahiro MoritaAffiliation: Department of Biochemistry and Goodman Cancer Research Centre, McGill University, Montreal, Canada
Bio-protocol author page: a708
Tommy Alain
Tommy AlainAffiliation: Department of Biochemistry and Goodman Cancer Research Centre, McGill University, Montreal, Canada
Bio-protocol author page: a709
Ivan Topisirovic
Ivan TopisirovicAffiliation: Department of Oncology and Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Montreal, Canada
For correspondence: ivan.topisirovic@mcgill.ca
Bio-protocol author page: a707
 and Nahum Sonenberg
Nahum SonenbergAffiliation: Department of Biochemistry and Goodman Cancer Research Centre, McGill University, Montreal, Canada
For correspondence: nahum.sonenberg@mcgill.ca
Bio-protocol author page: a710
date: 7/20/2013, 12962 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.833 

[Abstract] 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 ...

Article identifier: bio-protocol.org/e833

Plant Science

Analysis of Malondialdehyde, Chlorophyll Proline, Soluble Sugar, and Glutathione Content in Arabidopsis seedling

Authors: Zhijin Zhang
Zhijin ZhangAffiliation: Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou, China
Bio-protocol author page: a684
 and Rongfeng Huang
Rongfeng HuangAffiliation: Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou, China
For correspondence: rfhuang@caas.cn
Bio-protocol author page: a362
date: 7/20/2013, 11118 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.817 

[Abstract] 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 ...

Article identifier: bio-protocol.org/e817

Determination of Enzyme Kinetic Parameters of UDP-glycosyltransferases

Authors: Jörg M. Augustin
Jörg M. AugustinAffiliation: Faculty of Life Sciences - Department for Plant Biochemistry and Biotechnology, University of Copenhagen, Fredriksberg, Copenhagen, Denmark
For correspondence: jmaug@gmx.de
Bio-protocol author page: a403
 and Søren Bak
Søren BakAffiliation: Faculty of Life Sciences - Department for Plant Biochemistry and Biotechnology, University of Copenhagen, Fredriksberg, Copenhagen, Denmark
Bio-protocol author page: a404
date: 7/20/2013, 3776 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.825 

[Abstract] 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 ...

Article identifier: bio-protocol.org/e825

Extraction and Reglucosylation of Barbarea vulgaris Sapogenins

Authors: Jörg M. Augustin
Jörg M. AugustinAffiliation: Faculty of Life Sciences - Department for Plant Biochemistry and Biotechnology, University of Copenhagen, Fredriksberg, Copenhagen, Denmark
For correspondence: jmaug@gmx.de
Bio-protocol author page: a403
Carl Erik Olsen
Carl Erik OlsenAffiliation: Faculty of Life Sciences - Department of Basic Science and Environment, University of Copenhagen, Fredriksberg, Copenhagen, Denmark
Bio-protocol author page: a692
 and Søren Bak
Søren BakAffiliation: Faculty of Life Sciences - Department for Plant Biochemistry and Biotechnology, University of Copenhagen, Fredriksberg, Copenhagen, Denmark
Bio-protocol author page: a404
date: 7/20/2013, 3384 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.826 

[Abstract] 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 ...

Article identifier: bio-protocol.org/e826

Maize Endosperm Protein Extraction and Analysis

Authors: Xinze Chen
Xinze ChenAffiliation: School of Life Sciences, Shanghai University, Shanghai, China
Bio-protocol author page: a704
Dongsheng Yao
Dongsheng YaoAffiliation: School of Life Sciences, Shanghai University, Shanghai, China
Bio-protocol author page: a705
 and Rentao Song
Rentao SongAffiliation: School of Life Sciences, Shanghai University, Shanghai, China
For correspondence: rentaosong@staff.shu.edu.cn
Bio-protocol author page: a706
date: 7/20/2013, 3814 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.832 

[Abstract] 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. ...

Article identifier: bio-protocol.org/e832