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Biochemistry

Phos-tag Immunoblot Analysis for Detecting IRF5 Phosphorylation

Featured protocol,  Authors: Go R. Sato
Go R. SatoAffiliation: Department of Immunology, Yokohama City University Graduate School of Medicine, Yokohama, Japan
Bio-protocol author page: a4548
Tatsuma Ban
Tatsuma BanAffiliation: Department of Immunology, Yokohama City University Graduate School of Medicine, Yokohama, Japan
For correspondence: tatban@yokohama-cu.ac.jp
Bio-protocol author page: a4549
 and Tomohiko Tamura
Tomohiko TamuraAffiliation: Department of Immunology, Yokohama City University Graduate School of Medicine, Yokohama, Japan
For correspondence: tamurat@yokohama-cu.ac.jp
Bio-protocol author page: a4550
date: 5/20/2017, 126 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2295.

Brief version appeared in Immunity, Aug 2016
While the activation of the transcription factor interferon regulatory factor 5 (IRF5) is critical for the induction of innate immune responses, it also contributes to the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE). IRF5 phosphorylation is a hallmark of its activation in the Toll-like receptor (TLR) pathway, where active IRF5 induces type I interferon and proinflammatory cytokine genes. By using the phosphate-binding molecule Phos-tag, without either radioisotopes or phospho-specific antibodies, the protocol described here enables detection of the phosphorylation of both human and murine IRF5, as well as that of other proteins.

Spore Preparation Protocol for Enrichment of Clostridia from Murine Intestine

Featured protocol,  Authors: Eric M. Velazquez
Eric M. VelazquezAffiliation: Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA
Bio-protocol author page: a4551
Fabian Rivera-Chávez
Fabian Rivera-ChávezAffiliation: Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA
Bio-protocol author page: a4552
 and Andreas J. Bäumler
Andreas J. BäumlerAffiliation: Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA
For correspondence: ajbaumler@ucdavis.edu
Bio-protocol author page: a4553
date: 5/20/2017, 168 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2296.

Brief version appeared in Cell Host Microbe, Apr 2016
In recent years, many spore-forming commensal Clostridia found in the gut have been discovered to promote host physiology, immune development, and protection against infections. We provide a detailed protocol for rapid enrichment of spore-forming bacteria from murine intestine. Briefly, contents from the intestinal cecum are collected aerobically, diluted and finally treated with chloroform to enrich for Clostridia spores.

Analysis of in vivo Interaction between RNA Binding Proteins and Their RNA Targets by UV Cross-linking and Immunoprecipitation (CLIP) Method

Featured protocol,  Authors: Pamela Bielli
Pamela BielliAffiliation 1: Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
Affiliation 2: Laboratory of Neuroembriology, Fondazione Santa Lucia, Rome, Italy
Bio-protocol author page: a4492
 and Claudio Sette
Claudio SetteAffiliation 1: Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
Affiliation 2: Laboratory of Neuroembriology, Fondazione Santa Lucia, Rome, Italy
For correspondence: claudio.sette@uniroma2.it
Bio-protocol author page: a4493
date: 5/20/2017, 152 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2274.

Brief version appeared in Oncogene, Apr 2016
RNA metabolism is tightly controlled across different tissues and developmental stages, and its dysregulation is one of the molecular hallmarks of cancer. Through direct binding to specific sequence element(s), RNA binding proteins (RBPs) play a pivotal role in co- and post-transcriptional RNA regulatory events. We have recently demonstrated that, in pancreatic cancer cells, acquisition of a drug resistant (DR)-phenotype relied on upregulation of the polypyrimidine tract binding protein (PTBP1), which in turn is recruited to the pyruvate kinase pre-mRNA and favors splicing of the oncogenic PKM2 variant. Herein, we describe a step-by-step protocol of the ultraviolet (UV) light cross-linking and immunoprecipitation (CLIP) method to determine the direct binding of a RBP to specific regions of its target RNAs in adherent human cell lines.

Semi-quantitative Analysis of H4K20me1 Levels in Living Cells Using Mintbody

Featured protocol,  Authors: Yuko Sato
Yuko SatoAffiliation: Cell Biology Unit, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Japan
For correspondence: satoy@bio.titech.ac.jp
Bio-protocol author page: a4494
 and Hiroshi Kimura
Hiroshi KimuraAffiliation: Cell Biology Unit, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Japan
For correspondence: hkimura@bio.titech.ac.jp
Bio-protocol author page: a4495
date: 5/20/2017, 138 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2276.

Brief version appeared in J Mol Biol, Oct 2016
Eukaryotic nuclear DNA wraps around histone proteins to form a nucleosome, a basic unit of chromatin. Posttranslational modification of histones play an important role in gene regulation and chromosome duplication. Some modifications are quite stable to be an epigenetic memory, and others exhibit rapid turnover or fluctuate during the cell cycle. Histone H4 Lys20 monomethylation (H4K20me1) has been shown to be involved in chromosome condensation, segregation, replication and repair. H4K20 methylation is controlled through a few methyltransferases, PR-Set7/Set8, SUV420H1, and SUV420H2, and a demethylase, PHF8. In cycling cells, the level of H4K20me1 increases during G2 and M phases and decreases during G1 phase. To monitor the local concentration and global fluctuation of histone modifications in living cells, we have developed a genetically encoded probe termed mintbody (modification-specific intracellular antibody; Sato et al., 2013 and 2016). By measuring the nuclear to cytoplasmic intensity ratio, the relative level of H4K20me1 in individual cells can be monitored. This detailed protocol allows the semi-quantitative analysis of the effects of methyltransferases on H4K20me1 levels in living cells based on H4K20me1-mintbody described by Sato et al. (2016).

Protein Isolation from Plasma Membrane, Digestion and Processing for Strong Cation Exchange Fractionation

Featured protocol,  Authors: Ning Yang
Ning YangAffiliation: Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a4554
Bing Han
Bing HanAffiliation: Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a4555
 and Tai Wang
Tai WangAffiliation: Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China
For correspondence: twang@ibcas.ac.cn
Bio-protocol author page: a4558
date: 5/20/2017, 114 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2298.

Brief version appeared in J Integr Plant Biol, Dec 2010
Plasma membrane (PM) proteins play crucial roles in diverse biological processes. But their low abundance, alkalinity and hydrophobicity make their isolation a difficult task. This protocol describes an efficient method for PM proteins isolation, digestion and fractionation so that they can be well prepared for mass spectrometry analysis.

Assay to Measure Interactions between Purified Drp1 and Synthetic Liposomes

Featured protocol,  Authors: Yoshihiro Adachi
Yoshihiro AdachiAffiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, USA
Bio-protocol author page: a4472
Kie Itoh
Kie ItohAffiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, USA
Bio-protocol author page: a4473
Miho Iijima
Miho IijimaAffiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, USA
Bio-protocol author page: a4474
 and Hiromi Sesaki
Hiromi SesakiAffiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, USA
For correspondence: hsesaki@jhmi.edu
Bio-protocol author page: a4475
date: 5/5/2017, 245 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2266.

Brief version appeared in Mol Cell, Sep 2016
A mitochondrion is a dynamic intracellular organelle that actively divides and fuses to control its size, number and shape in cells. A regulated balance between mitochondrial division and fusion is fundamental to the function, distribution and turnover of mitochondria (Roy et al., 2015). Mitochondrial division is mediated by dynamin-related protein 1 (Drp1), a mechano-chemical GTPase that constricts mitochondrial membranes (Tamura et al., 2011). Mitochondrial membrane lipids such as phosphatidic acid and cardiolipin bind Drp1, and Drp1-phospholipid interactions provide key regulatory mechanisms for mitochondrial division (Montessuit et al., 2010; Bustillo-Zabalbeitia et al., 2014; Macdonald et al., 2014; Stepanyants et al., 2015; Adachi et al., 2016). Here, we describe biochemical experiments that quantitatively measure interactions of Drp1 with lipids using purified recombinant Drp1 and synthetic liposomes with a defined set of phospholipids. This assay makes it possible to define the specificity of protein-lipid interaction and the role of the head group and acyl chains.

Mating Based Split-ubiquitin Assay for Detection of Protein Interactions

Featured protocol,  Authors: Wijitra Horaruang
Wijitra HoraruangAffiliation: Laboratory of Plant Physiology and Biophysics, Bower Building, University of Glasgow, Glasgow, UK
Bio-protocol author page: a4456
 and Ben Zhang
Ben ZhangAffiliation: Laboratory of Plant Physiology and Biophysics, Bower Building, University of Glasgow, Glasgow, UK
For correspondence: Ben.Zhang@glasgow.ac.uk
Bio-protocol author page: a4457
date: 5/5/2017, 312 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2258.

Brief version appeared in Plant Cell, Jun 2015
The mating based split-ubiquitin (mbSUS) assay is an alternative method to the classical yeast two-hybrid system with a number of advantages. The mbSUS assay relies on the ubiquitin-degradation pathway as a sensor for protein-protein interactions, and it is suitable for the determination of interactions between full-length proteins that are cytosolic or membrane-bound. Here we describe the mbSUS assay protocol which has been used for detecting the interaction between K+ channel and SNARE proteins (Grefen et al., 2010 and 2015; Zhang et al., 2015 and 2016)

Biotinylated Micro-RNA Pull Down Assay for Identifying miRNA Targets

Featured protocol,  Authors: Pornima Phatak
Pornima PhatakAffiliation 1: Department of Surgery, University of Maryland School of Medicine, Baltimore, USA
Affiliation 2: Baltimore Veterans Affairs Medical Center, Baltimore, USA
Bio-protocol author page: a4420
 and James M Donahue
James M DonahueAffiliation 1: Department of Surgery, University of Maryland School of Medicine, Baltimore, USA
Affiliation 2: Baltimore Veterans Affairs Medical Center, Baltimore, USA
For correspondence: jdonahue@som.umaryland.edu
Bio-protocol author page: a4421
date: 5/5/2017, 202 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2253.

Brief version appeared in Oncogene, Apr 2016
microRNA (miRNA) directly associates with its target transcripts (mRNA). This protocol describes a method for detection of direct interaction between miRNA and mRNA. The result of interaction helps screening the specific target mRNAs for a miRNA.

Measuring Cyanobacterial Metabolism in Biofilms with NanoSIMS Isotope Imaging and Scanning Electron Microscopy (SEM)

Featured protocol,  Authors: Rhona K. Stuart
Rhona K. StuartAffiliation: Physical and Life Sciences Division, Lawrence Livermore National Laboratory, Livermore, USA
For correspondence: stuart25@llnl.gov
Bio-protocol author page: a4466
Xavier Mayali
Xavier MayaliAffiliation: Physical and Life Sciences Division, Lawrence Livermore National Laboratory, Livermore, USA
Bio-protocol author page: a4467
Michael P. Thelen
Michael P. ThelenAffiliation: Physical and Life Sciences Division, Lawrence Livermore National Laboratory, Livermore, USA
Bio-protocol author page: a4468
Jennifer Pett-Ridge
Jennifer Pett-RidgeAffiliation: Physical and Life Sciences Division, Lawrence Livermore National Laboratory, Livermore, USA
Bio-protocol author page: a4469
 and Peter K. Weber
Peter K. WeberAffiliation: Physical and Life Sciences Division, Lawrence Livermore National Laboratory, Livermore, USA
Bio-protocol author page: a4470
date: 5/5/2017, 168 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2263.

Brief version appeared in Mbio, Jun 2016
To advance the understanding of microbial interactions, it is becoming increasingly important to resolve the individual metabolic contributions of microorganisms in complex communities. Organisms from biofilms can be especially difficult to separate, image and analyze, and methods to address these limitations are needed. High resolution imaging secondary ion mass spectrometry (NanoSIMS) generates single cell isotopic composition measurements, and can be used to quantify incorporation and exchange of an isotopically labeled substrate among individual organisms. Here, incorporation of cyanobacterial extracellular organic matter (EOM) by members of a cyanobacterial mixed species biofilm is used as a model to illustrate this method. Incorporation of stable isotope labeled (15N and 13C) EOM by two groups, cyanobacteria and associated heterotrophic microbes, are quantified. Methods for generating, preparing, and analyzing samples for quantifying uptake of stable isotope-labeled EOM in the biofilm are described.

Incubation of Cyanobacteria under Dark, Anaerobic Conditions and Quantification of the Excreted Organic Acids by HPLC

Featured protocol,  Authors: Chika Yasuda
Chika YasudaAffiliation: School of Agriculture, Meiji University, Kanagawa, Japan
Bio-protocol author page: a4452
Hiroko Iijima
Hiroko IijimaAffiliation: School of Agriculture, Meiji University, Kanagawa, Japan
Bio-protocol author page: a4453
Haruna Sukigara
Haruna SukigaraAffiliation: School of Agriculture, Meiji University, Kanagawa, Japan
Bio-protocol author page: a4454
 and Takashi Osanai
Takashi OsanaiAffiliation: School of Agriculture, Meiji University, Kanagawa, Japan
For correspondence: tosanai@meiji.ac.jp
Bio-protocol author page: a4455
date: 5/5/2017, 146 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2257.

Brief version appeared in Sci Rep, Aug 2016
Succinate and lactate are commodity chemicals used for producing bioplastics. Recently, it was found that such organic acids are excreted from cells of the unicellular cyanobacterium Synechocystis sp. PCC 6803 under dark, anaerobic conditions. To conduct the dark, anaerobic incubation, cells were concentrated within a vial that was then sealed with a butyl rubber cap, following which N2 gas was introduced into the vial. The organic acids produced were quantified by high-performance liquid chromatography via post-labeling with bromothymol blue as a pH indicator. After separation by ion-exclusion chromatography, the organic acids were identified by comparing their retention time with that of standard solutions. These procedures allow researchers to quantify the organic acids produced by microorganisms, contributing to knowledge about the biology and biotechnology of cyanobacteria.

Metabolic Heavy Isotope Labeling to Study Glycerophospholipid Homeostasis of Cultured Cells

Featured protocol,  Authors: Satu Hänninen
Satu HänninenAffiliation: Department of Biochemistry and Developmental Biology, Medical Faculty, University of Helsinki, Helsinki, Finland
For correspondence: satu.m.hanninen@helsinki.fi
Bio-protocol author page: a4478
Pentti Somerharju
Pentti SomerharjuAffiliation: Department of Biochemistry and Developmental Biology, Medical Faculty, University of Helsinki, Helsinki, Finland
Bio-protocol author page: a4479
 and Martin Hermansson
Martin HermanssonAffiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark
Bio-protocol author page: a4480
date: 5/5/2017, 163 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2268.

Brief version appeared in Biochim Biophys Acta, Sep 2016
Glycerophospholipids consist of a glycerophosphate backbone to which are esterified two acyl chains and a polar head group. The head group (e.g., choline, ethanolamine, serine or inositol) defines the glycerophospholipid class, while the acyl chains together with the head group define the glycerophospholipid molecular species. Stable heavy isotope (e.g., deuterium)-labeled head group precursors added to the culture medium incorporate efficiently into glycerophospholipids of mammalian cells, which allows one to determine the rates of synthesis, acyl chain remodeling or turnover of the individual glycerophospholipids using mass spectrometry. This protocol describes how to study the metabolism of the major mammalian glycerophospholipids i.e., phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines and phosphatidylinositols with this approach.

Preparation of Everted Membrane Vesicles from Escherichia coli Cells

Featured protocol,  Author: Marina Verkhovskaya
Marina VerkhovskayaAffiliation: Institute of Biotechnology, PO Box 65 (Viikinkaari 1) FIN-00014 University of Helsinki, Helsinki, Finland
For correspondence: Marina.Verkhovskaya@Helsinki.Fi
Bio-protocol author page: a4446
date: 5/5/2017, 147 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2254.

Brief version appeared in FEBS Lett, Jun 2016
The protocol for obtaining electrically sealed membrane vesicles from E. coli cells is presented. Proton pumps such as Complex I, quinol oxidase, and ATPase are active in the obtained vesicles. Quality of the preparation was tested by monitoring the electric potential generated by these pumps.

Immunoprecipitation of Cell Surface Proteins from Gram-negative Bacteria

Featured protocol,  Authors: Carlos Eduardo Pouey Cunha*
Carlos Eduardo Pouey CunhaAffiliation 1: School of Biosciences and Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK
Affiliation 2: Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, Rio Grande do Sul, Brasil
Bio-protocol author page: a4431
Jane Newcombe*
Jane NewcombeAffiliation: School of Biosciences and Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK
Bio-protocol author page: a4432
Odir Antonio Dellagostin
Odir Antonio DellagostinAffiliation: Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, Rio Grande do Sul, Brasil
Bio-protocol author page: a4433
 and Johnjoe McFadden
Johnjoe McFaddenAffiliation: School of Biosciences and Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK
For correspondence: j.mcfadden@surrey.ac.uk
Bio-protocol author page: a4434
 (*contributed equally to this work) date: 5/5/2017, 199 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2250.

Brief version appeared in Microbiology, Feb 2014
The meningococcus (Neisseria meningitidis) remains an important threat to human health worldwide. This Gram-negative bacterium causes elevated disabilities and mortality in infected individuals. Despite several available vaccines, currently there is no universal vaccine against all circulating meningococcal strains (Vogel et al., 2013). Herein, we describe a new protocol that is capable of identifying only cell surface exposed proteins that play a role in immunity, providing this research field with a more straightforward approach to identify novel vaccine targets. Even though N. meningitidis is used as a model in the protocol herein described, this protocol can be used for any Gram-negative bacteria provided modifications and optimizations are carried out to adapt it to different bacterial and disease characteristics (e.g., membrane fragility, growth methods, serum antibody levels, etc.).

Phos-tag Immunoblot Analysis for Detecting IRF5 Phosphorylation

Authors: Go R. Sato
Go R. SatoAffiliation: Department of Immunology, Yokohama City University Graduate School of Medicine, Yokohama, Japan
Bio-protocol author page: a4548
Tatsuma Ban
Tatsuma BanAffiliation: Department of Immunology, Yokohama City University Graduate School of Medicine, Yokohama, Japan
For correspondence: tatban@yokohama-cu.ac.jp
Bio-protocol author page: a4549
 and Tomohiko Tamura
Tomohiko TamuraAffiliation: Department of Immunology, Yokohama City University Graduate School of Medicine, Yokohama, Japan
For correspondence: tamurat@yokohama-cu.ac.jp
Bio-protocol author page: a4550
date: 5/20/2017, 126 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2295.

[Abstract] While the activation of the transcription factor interferon regulatory factor 5 (IRF5) is critical for the induction of innate immune responses, it also contributes to the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE). IRF5 phosphorylation is a hallmark of its activation in the Toll-like receptor (TLR) pathway, where active ...

Spore Preparation Protocol for Enrichment of Clostridia from Murine Intestine

Authors: Eric M. Velazquez
Eric M. VelazquezAffiliation: Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA
Bio-protocol author page: a4551
Fabian Rivera-Chávez
Fabian Rivera-ChávezAffiliation: Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA
Bio-protocol author page: a4552
 and Andreas J. Bäumler
Andreas J. BäumlerAffiliation: Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA
For correspondence: ajbaumler@ucdavis.edu
Bio-protocol author page: a4553
date: 5/20/2017, 168 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2296.

[Abstract] In recent years, many spore-forming commensal Clostridia found in the gut have been discovered to promote host physiology, immune development, and protection against infections. We provide a detailed protocol for rapid enrichment of spore-forming bacteria from murine intestine. Briefly, contents from the intestinal cecum are collected aerobically, ...

Analysis of in vivo Interaction between RNA Binding Proteins and Their RNA Targets by UV Cross-linking and Immunoprecipitation (CLIP) Method

Authors: Pamela Bielli
Pamela BielliAffiliation 1: Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
Affiliation 2: Laboratory of Neuroembriology, Fondazione Santa Lucia, Rome, Italy
Bio-protocol author page: a4492
 and Claudio Sette
Claudio SetteAffiliation 1: Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
Affiliation 2: Laboratory of Neuroembriology, Fondazione Santa Lucia, Rome, Italy
For correspondence: claudio.sette@uniroma2.it
Bio-protocol author page: a4493
date: 5/20/2017, 152 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2274.

[Abstract] RNA metabolism is tightly controlled across different tissues and developmental stages, and its dysregulation is one of the molecular hallmarks of cancer. Through direct binding to specific sequence element(s), RNA binding proteins (RBPs) play a pivotal role in co- and post-transcriptional RNA regulatory events. We have recently demonstrated that, ...

Semi-quantitative Analysis of H4K20me1 Levels in Living Cells Using Mintbody

Authors: Yuko Sato
Yuko SatoAffiliation: Cell Biology Unit, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Japan
For correspondence: satoy@bio.titech.ac.jp
Bio-protocol author page: a4494
 and Hiroshi Kimura
Hiroshi KimuraAffiliation: Cell Biology Unit, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Japan
For correspondence: hkimura@bio.titech.ac.jp
Bio-protocol author page: a4495
date: 5/20/2017, 138 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2276.

[Abstract] Eukaryotic nuclear DNA wraps around histone proteins to form a nucleosome, a basic unit of chromatin. Posttranslational modification of histones play an important role in gene regulation and chromosome duplication. Some modifications are quite stable to be an epigenetic memory, and others exhibit rapid turnover or fluctuate during the cell cycle. Histone ...

Protein Isolation from Plasma Membrane, Digestion and Processing for Strong Cation Exchange Fractionation

Authors: Ning Yang
Ning YangAffiliation: Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a4554
Bing Han
Bing HanAffiliation: Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a4555
 and Tai Wang
Tai WangAffiliation: Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China
For correspondence: twang@ibcas.ac.cn
Bio-protocol author page: a4558
date: 5/20/2017, 114 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2298.

[Abstract] Plasma membrane (PM) proteins play crucial roles in diverse biological processes. But their low abundance, alkalinity and hydrophobicity make their isolation a difficult task. This protocol describes an efficient method for PM proteins isolation, digestion and fractionation so that they can be well prepared for mass spectrometry analysis....

Assay to Measure Interactions between Purified Drp1 and Synthetic Liposomes

Authors: Yoshihiro Adachi
Yoshihiro AdachiAffiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, USA
Bio-protocol author page: a4472
Kie Itoh
Kie ItohAffiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, USA
Bio-protocol author page: a4473
Miho Iijima
Miho IijimaAffiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, USA
Bio-protocol author page: a4474
 and Hiromi Sesaki
Hiromi SesakiAffiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, USA
For correspondence: hsesaki@jhmi.edu
Bio-protocol author page: a4475
date: 5/5/2017, 245 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2266.

[Abstract] A mitochondrion is a dynamic intracellular organelle that actively divides and fuses to control its size, number and shape in cells. A regulated balance between mitochondrial division and fusion is fundamental to the function, distribution and turnover of mitochondria (Roy et al., 2015). Mitochondrial division is mediated by dynamin-related protein ...

Mating Based Split-ubiquitin Assay for Detection of Protein Interactions

Authors: Wijitra Horaruang
Wijitra HoraruangAffiliation: Laboratory of Plant Physiology and Biophysics, Bower Building, University of Glasgow, Glasgow, UK
Bio-protocol author page: a4456
 and Ben Zhang
Ben ZhangAffiliation: Laboratory of Plant Physiology and Biophysics, Bower Building, University of Glasgow, Glasgow, UK
For correspondence: Ben.Zhang@glasgow.ac.uk
Bio-protocol author page: a4457
date: 5/5/2017, 312 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2258.

[Abstract] The mating based split-ubiquitin (mbSUS) assay is an alternative method to the classical yeast two-hybrid system with a number of advantages. The mbSUS assay relies on the ubiquitin-degradation pathway as a sensor for protein-protein interactions, and it is suitable for the determination of interactions between full-length proteins that are cytosolic ...

Biotinylated Micro-RNA Pull Down Assay for Identifying miRNA Targets

Authors: Pornima Phatak
Pornima PhatakAffiliation 1: Department of Surgery, University of Maryland School of Medicine, Baltimore, USA
Affiliation 2: Baltimore Veterans Affairs Medical Center, Baltimore, USA
Bio-protocol author page: a4420
 and James M Donahue
James M DonahueAffiliation 1: Department of Surgery, University of Maryland School of Medicine, Baltimore, USA
Affiliation 2: Baltimore Veterans Affairs Medical Center, Baltimore, USA
For correspondence: jdonahue@som.umaryland.edu
Bio-protocol author page: a4421
date: 5/5/2017, 202 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2253.

[Abstract] microRNA (miRNA) directly associates with its target transcripts (mRNA). This protocol describes a method for detection of direct interaction between miRNA and mRNA. The result of interaction helps screening the specific target mRNAs for a miRNA. ...

Measuring Cyanobacterial Metabolism in Biofilms with NanoSIMS Isotope Imaging and Scanning Electron Microscopy (SEM)

Authors: Rhona K. Stuart
Rhona K. StuartAffiliation: Physical and Life Sciences Division, Lawrence Livermore National Laboratory, Livermore, USA
For correspondence: stuart25@llnl.gov
Bio-protocol author page: a4466
Xavier Mayali
Xavier MayaliAffiliation: Physical and Life Sciences Division, Lawrence Livermore National Laboratory, Livermore, USA
Bio-protocol author page: a4467
Michael P. Thelen
Michael P. ThelenAffiliation: Physical and Life Sciences Division, Lawrence Livermore National Laboratory, Livermore, USA
Bio-protocol author page: a4468
Jennifer Pett-Ridge
Jennifer Pett-RidgeAffiliation: Physical and Life Sciences Division, Lawrence Livermore National Laboratory, Livermore, USA
Bio-protocol author page: a4469
 and Peter K. Weber
Peter K. WeberAffiliation: Physical and Life Sciences Division, Lawrence Livermore National Laboratory, Livermore, USA
Bio-protocol author page: a4470
date: 5/5/2017, 168 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2263.

[Abstract] To advance the understanding of microbial interactions, it is becoming increasingly important to resolve the individual metabolic contributions of microorganisms in complex communities. Organisms from biofilms can be especially difficult to separate, image and analyze, and methods to address these limitations are needed. High resolution imaging secondary ...

Incubation of Cyanobacteria under Dark, Anaerobic Conditions and Quantification of the Excreted Organic Acids by HPLC

Authors: Chika Yasuda
Chika YasudaAffiliation: School of Agriculture, Meiji University, Kanagawa, Japan
Bio-protocol author page: a4452
Hiroko Iijima
Hiroko IijimaAffiliation: School of Agriculture, Meiji University, Kanagawa, Japan
Bio-protocol author page: a4453
Haruna Sukigara
Haruna SukigaraAffiliation: School of Agriculture, Meiji University, Kanagawa, Japan
Bio-protocol author page: a4454
 and Takashi Osanai
Takashi OsanaiAffiliation: School of Agriculture, Meiji University, Kanagawa, Japan
For correspondence: tosanai@meiji.ac.jp
Bio-protocol author page: a4455
date: 5/5/2017, 146 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2257.

[Abstract] Succinate and lactate are commodity chemicals used for producing bioplastics. Recently, it was found that such organic acids are excreted from cells of the unicellular cyanobacterium Synechocystis sp. PCC 6803 under dark, anaerobic conditions. To conduct the dark, anaerobic incubation, cells were concentrated within a vial that was then sealed with ...
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[Bio101] Bradford Protein Assay

Author: Fanglian He date: 3/20/2011, 92927 views, 6 Q&A
DOI: https://doi.org/10.21769/BioProtoc.45.

[Abstract] The Bradford protein assay is used to measure the concentration of total protein in a sample. The principle of this assay is that the binding of protein molecules to Coomassie dye under acidic conditions results in a color change from brown to blue. This method actually measures the presence of the ...

[Bio101] Laemmli-SDS-PAGE

Author: Fanglian He date: 6/5/2011, 67039 views, 10 Q&A
DOI: https://doi.org/10.21769/BioProtoc.80.

[Abstract] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is used to separate proteins with relative molecular mass no smaller than 10 KD. Very small proteins (<10 KD) are difficult to resolve due to low ability of binding to SDS, which can be solved by gradient gels or using different eletrophoresis ...

Detection of Intracellular Reactive Oxygen Species (CM-H2DCFDA)

Authors: Rabii Ameziane-El-Hassani
Rabii Ameziane-El-HassaniAffiliation 1: UBRM, Centre National de l'Energie, des Sciences et des Techniques Nucléaires, Rabat, Morocco
Affiliation 2: Institut Gustave Roussy, FRE2939 Centre National de la Recherche Scientifique, Villejuif, France
Bio-protocol author page: a200
 and Corinne Dupuy
Corinne DupuyAffiliation 1: Institut Gustave Roussy, FRE2939 Centre National de la Recherche Scientifique, Villejuif, France
Affiliation 2: University Paris, Sud 11, Orsay, France
For correspondence: dupuy@igr.fr
Bio-protocol author page: a201
date: 1/5/2013, 42366 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.313.

[Abstract] Reactive oxygen species (ROS) play a critical role in cellular physiopathology. ROS are implicated in cell proliferation, signaling pathways, oxidative defense mechanisms responsible for killing of bacteria, thyroid hormonosynthesis, etc. The cellular Redox homeostasis is balanced by oxidants and antioxidants ...

[Bio101] GST-Pull Down Protocol

Author: Lili Jing
Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
date: 1/20/2012, 41875 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.177.

[Abstract] GST-Pull down assay is an effective way to examine the direct binding of two proteins in vitro. This protocol is based on GST pull down system from GE healthcare, and uses the binding of unplugged/MuSK receptor and Wnt ligand as an example to illustrate the detailed procedure....

[Bio101] BCA (Bicinchoninic Acid) Protein Assay

Author: Fanglian He
Fanglian HeAffiliation: Department of Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: fhe@bio-protocol.org
Bio-protocol author page: a9
date: 3/5/2011, 34966 views, 3 Q&A
DOI: https://doi.org/10.21769/BioProtoc.44.

[Abstract] The BCA protein assay is used for quantitation of total protein in a sample. The principle of this method is that proteins can reduce Cu+2 to Cu+1 in an alkaline solution (the biuret reaction) and result in a purple color formation by bicinchoninic acid. The reduction of copper is mainly caused by four ...

Detection of Hydrogen Peroxide by DAB Staining in Arabidopsis Leaves Updates
The author made some updates (highlighted in blue) to the protocol on 09/19/2016.

Authors: Arsalan Daudi
Arsalan DaudiAffiliation 1: Department of Biological Sciences, Royal Holloway University of London, Egham, UK
Affiliation 2: Department of Plant Pathology, University of California, Davis, CA, USA
For correspondence: aadaudi@ucdavis.edu
Bio-protocol author page: a107
 and Jose A. O’Brien
Jose A. O’BrienAffiliation: Department of Biological Sciences, Royal Holloway University of London, Egham, UK
Bio-protocol author page: a108
date: 9/20/2012, 33156 views, 16 Q&A
DOI: https://doi.org/10.21769/BioProtoc.263.

[Abstract] In this protocol, the in situ detection of hydrogen peroxide (one of several reactive oxygen species) is described in mature Arabidopsis rosette leaves by staining with 3,3'-diaminobenzidine (DAB) using an adaptation of previous methods (Thordal-Christensen et al., 1997; Bindschedler et al., 2006; Daudi ...

[Bio101] Infiltration of Nicotiana benthamiana Protocol for Transient Expression via Agrobacterium

Author: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
date: 7/20/2011, 31987 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.95.

[Abstract] Transient expression in tobacco plant (Nicotiana benthamiana) is used to determine the subcellular location of a protein of interest when tagged with a reporter such as green fluorescent protein (GFP), or to mass produce proteins without making transgenic plants. The root tumor bacteria, Agrobacteria, ...

[Bio101] Coomassie Blue Staining

Author: Fanglian He date: 6/5/2011, 31222 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.78.

[Abstract] Coomassie staining is able to detect protein bands containing about 0.2 μg or more protein. For low abundant protein, silver staining (www/silver staining) is a better choice since it is about 100-fold more sensitive than Coomassie staining....

[Bio101] Glucose Tolerance Test in Mice

Author: Peichuan Zhang
Peichuan ZhangAffiliation 1: Department of Biology, The Pennsylvania State University, University Park, PA, USA
Affiliation 2: Department of Biochemistry and Biophysics, University of California, San Francisco, USA
For correspondence: peichuan.zhang@ucsf.edu
Bio-protocol author page: a11
date: 10/5/2011, 30838 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.159.

[Abstract] Glucose tolerance test is a standard procedure that addresses how quickly exogenous glucose can be cleared from blood. Specifically, uptake of glucose from the blood by cells is regulated by insulin. Impairment of glucose tolerance (i.e, longer time to clear given amount of glucose) indicates problems ...

[Bio101] A General EMSA (Gel-shift) Protocol

Author: Ran Chen
Ran ChenAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: rcchen@jfkbio.com
Bio-protocol author page: a34
date: 2/5/2011, 25207 views, 3 Q&A
DOI: https://doi.org/10.21769/BioProtoc.24.

[Abstract] An electrophoretic mobility shift assay (EMSA), also referred to as mobility shift electrophoresis, a gel shift assay, gel mobility shift assay, band shift assay, or gel retardation assay, is a common technique used to study protein-DNA or protein-RNA interactions. The control lane (the DNA/RNA probe ...
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