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Biochemistry

Polysome Analysis

Featured protocol,  Authors: Dipak Kumar Poria
Dipak Kumar PoriaAffiliation: Department of Biological Sciences, Indian Institute of Science Education and Research, Kolkata, India
Bio-protocol author page: a4215
 and Partho Sarothi Ray
Partho Sarothi RayAffiliation: Department of Biological Sciences, Indian Institute of Science Education and Research, Kolkata, India
For correspondence: psray@iiserkol.ac.in
Bio-protocol author page: a4216
date: 3/20/2017, 144 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2192.

Brief version appeared in Oncogene, Mar 2016
Polysome analysis is a method to separate mRNAs from a cell into actively translating and non-translating fractions depending on their association with polysomes. By this protocol, cell lysates are fractionated by sucrose density gradient ultracentrifugation. Free mRNA fraction and various ribosomal fractions, such as 40S, 60S, monosomes and polysomes are collected by fractionation. Association of particular mRNAs with these fractions is detected by reverse transcription – PCR to investigate the translational state of the mRNA.

Measurement of RNA-induced PKR Activation in vitro

Featured protocol,  Author: Kobe C. Yuen
Kobe C. YuenAffiliation: Stowers Institute for Medical Research, Kansas City, MO, USA
Present address: 1 DNA Way, South San Francisco, USA
For correspondence: yuenc4@gene.com
Bio-protocol author page: a4260
date: 3/20/2017, 141 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2178.

Brief version appeared in Cell Rep, Jan 2016
Protein kinase R (PKR) is one of the key RNA-activated sensors for innate immunity. PKR is activated by pathogenic or aberrant RNAs such as short double-stranded RNAs or those with imperfect secondary structures, as well as a reduction in the amount and number of RNA modifications. Activation of PKR may be an underlying mechanism for the pathogenesis of human diseases. In this protocol, I describe a method for studying levels of RNA-induced PKR activation in vitro.

Chase Assay of Protein Stability in Haloferax volcanii

Featured protocol,  Authors: Xian Fu
Xian FuAffiliation: Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, USA
Bio-protocol author page: a4266
 and Julie A. Maupin-Furlow
Julie A. Maupin-FurlowAffiliation 1: Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, USA
Affiliation 2: Genetics Institute, University of Florida, Gainesville, Florida, USA
For correspondence: jmaupin@ufl.edu
Bio-protocol author page: a4267
date: 3/20/2017, 107 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2186.

Brief version appeared in mBio, May 2016
Highly regulated and targeted protein degradation plays a fundamental role in almost all cellular processes. Determination of the protein half-life by the chase assay serves as a powerful and popular strategy to compare the protein stability and study proteolysis pathways in cells. Here, we describe a chase assay in Haloferax volcanii, a halophilic archaeon as the model organism.

Rubisco Extraction and Purification from Diatoms

Featured protocol,  Authors: Jodi N. Young
Jodi N. YoungAffiliation: Department of Oceanography, University of Washington, Seattle, USA
For correspondence: youngjn@uw.edu
Bio-protocol author page: a4026
Ana M. C. Heureux
Ana M. C. HeureuxAffiliation: Department of Earth Sciences, University of Oxford, Oxford, UK
Bio-protocol author page: a4027
Rosalind E. M. Rickaby
Rosalind E. M. RickabyAffiliation: Department of Earth Sciences, University of Oxford, Oxford, UK
Bio-protocol author page: a4028
François M. M. Morel
François M. M. MorelAffiliation: Department of Geosciences, Princeton University, Princeton, USA
Bio-protocol author page: a4029
Spencer M. Whitney
Spencer M. WhitneyAffiliation: Plant Science Division, Research School of Biology, the Australian National University, Canberra, Australia
Bio-protocol author page: a4030
 and Robert E. Sharwood
Robert E. SharwoodAffiliation: Plant Science Division, Research School of Biology, the Australian National University, Canberra, Australia
Bio-protocol author page: a4031
date: 3/20/2017, 134 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2191.

Brief version appeared in J Exp Bot, May 2016
This protocol describes a method to extract ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) from diatoms (Bacillariophyta) to determine catalytic performance. This protocol has been adapted from use in cyanobacteria and higher plants (Andrews, 1988; Whitney and Sharwood, 2007). First part (steps A1-A3) of the extraction provides a crude extract of Rubisco that is sufficient for carboxylation assays to measure the Michaelis constant for CO2 (KC) and the catalytic turnover rate (kcatc). However, the further purification steps outlined (steps B1-B4) are needed for measurements of Rubisco CO2/O2 Specificity (SC/O, [Kane et al., 1994]).

Determination of Adeno-associated Virus Rep DNA Binding Using Fluorescence Anisotropy

Featured protocol,  Authors: Francisco Zarate-Perez
Francisco Zarate-PerezAffiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond VA, USA
Bio-protocol author page: a4284
Vishaka Santosh
Vishaka SantoshAffiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond VA, USA
Bio-protocol author page: a4286
Martino Bardelli
Martino BardelliAffiliation: Department of Infectious Diseases, King’s College London, London, United Kingdom
Bio-protocol author page: a4283
Leticia Agundez
Leticia AgundezAffiliation: Department of Infectious Diseases, King’s College London, London, United Kingdom
Bio-protocol author page: a4285
R. Michael Linden
R. Michael LindenAffiliation: Department of Infectious Diseases, King’s College London, London, United Kingdom
Bio-protocol author page: a4287
Els Henckaerts
Els HenckaertsAffiliation: Department of Infectious Diseases, King’s College London, London, United Kingdom
Bio-protocol author page: a4289
 and Carlos R. Escalante
Carlos R. EscalanteAffiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond VA, USA
For correspondence: carlos.escalante@vcuhealth.org
Bio-protocol author page: a4288
date: 3/20/2017, 93 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2194.

Brief version appeared in J Virol, Jul 2016
Quantitative measurement of proteins binding to DNA is a requisite to fully characterize the structural determinants of complex formation necessary to understand the DNA transactions that regulate cellular processes. Here we describe a detailed protocol to measure binding affinity of the adeno-associated virus (AAV) Rep68 protein for the integration site AAVS1 using fluorescent anisotropy. This protocol can be used to measure the binding constants of any DNA binding protein provided the substrate DNA is fluorescently labeled.

Extraction, Purification and Quantification of Diffusible Signal Factor Family Quorum-sensing Signal Molecules in Xanthomonas oryzae pv. oryzae

Featured protocol,  Authors: Lian Zhou
Lian ZhouAffiliation 1: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Affiliation 2: Zhiyuan Innovative Research Center, Shanghai Jiao Tong University, Shanghai, China
Bio-protocol author page: a4225
Xing-Yu Wang
Xing-Yu WangAffiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Bio-protocol author page: a4226
Wei Zhang
Wei ZhangAffiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Bio-protocol author page: a4227
Shuang Sun
Shuang SunAffiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Bio-protocol author page: a4228
 and Ya-Wen He
Ya-Wen HeAffiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
For correspondence: yawenhe@sjtu.edu.cn
Bio-protocol author page: a4229
date: 3/20/2017, 108 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2190.

Brief version appeared in Mol Plant Microbe Interact, Mar 2016
Bacteria use quorum-sensing (QS) systems to monitor and regulate their population density. Bacterial QS involves small molecules that act as signals for bacterial communication. Many Gram-negative bacterial pathogens use a class of widely conserved molecules, called diffusible signal factor (DSF) family QS signals. The measurement of DSF family signal molecules is essential for understanding DSF metabolic pathways, signaling networks, as well as regulatory roles. Here, we describe a method for the extraction of DSF family signal molecules from Xanthomonas oryzae pv. oryzae (Xoo) cell pellets and Xoo culture supernatant. We determined the levels of DSF family signals using ultra-performance liquid chromatographic system (UPLC) coupled with accurate mass time-of-flight mass spectrometer (TOF-MS). With the aid of UPLC/MS system, the detection limit of DSF was as low as 1 µM, which greatly improves the ability to detect DSF DSF family signal molecules in bacterial cultures and reaction mixtures.

Isolation of Ribosomal Particles from the Unicellular Cyanobacterium Synechocystis sp. PCC 6803

Featured protocol,  Authors: Carla V. Galmozzi
Carla V. GalmozziAffiliation 1: Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla, Sevilla, Spain
Affiliation 2: Zentrum für Molekulare Biologie der Universität Heidelberg, Heidelberg, Germany
Bio-protocol author page: a4172
 and M. Isabel Muro-Pastor
M. Isabel Muro-Pastor Affiliation: Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla, Sevilla, Spain
For correspondence: imuro@ibvf.csic.es
Bio-protocol author page: a4173
date: 3/20/2017, 107 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2176.

Brief version appeared in PLoS One, Jul 2016
Isolation of ribosomal particles is an essential step in the study of ribosomal components as well as in the analysis of trans-acting factors that interact with the ribosome to regulate protein synthesis and modulate the expression profile of the cell in response to different environmental conditions. In this protocol, we describe a procedure for the isolation of 70S ribosomes from the unicellular cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). We have successfully used this protocol in our study of the cyanobacterial ribosomal-associated protein LrtA, which is a homologue of bacterial HPF (hibernation promoting factor) (Galmozzi et al., 2016).

Assays for the Detection of Rubber Oxygenase Activities

Featured protocol,  Authors: Wolf Röther
Wolf RötherAffiliation: Institute of Microbiology, University Stuttgart, Stuttgart, Germany
Bio-protocol author page: a4273
Jakob Birke
Jakob BirkeAffiliation: Institute of Microbiology, University Stuttgart, Stuttgart, Germany
Bio-protocol author page: a4274
 and Dieter Jendrossek
Dieter JendrossekAffiliation: Institute of Microbiology, University Stuttgart, Stuttgart, Germany
For correspondence: dieter.jendrossek@imb.uni-stuttgart.de
Bio-protocol author page: a4275
date: 3/20/2017, 117 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2188.

Brief version appeared in BMC Microbiol, May 2016
Microbial biodegradation of rubber relies on extracellular rubber oxygenases that catalyze the oxidative cleavage of the double bond of the polyisoprene backbone into oligo-isoprenoids. This protocol describes the determination of rubber oxygenase activities by an online measurement of molecular oxygen consumption via a non-invasive fluorescence-based assay. The produced oligo-isoprenoid cleavage products with terminal keto- and aldehyde-groups are identified qualitatively and quantitatively by HPLC. Our method allows for the characterization of homologue rubber oxygenases, and can likely be adapted to assay other oxygenases consuming dioxygen. Here we describe the determination of rubber oxygenase activities at the examples of the so far two known types of rubber oxygenases, namely rubber oxygenase A (RoxA) and latex clearing protein (Lcp).

Protein Synthesis Rate Assessment by Fluorescence Recovery after Photobleaching (FRAP)

Featured protocol,  Authors: Nikos Kourtis
Nikos KourtisAffiliation 1: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Crete, Greece
Affiliation 2: Department of Pathology, NYU School of Medicine, New York, USA
Bio-protocol author page: a4141
 and Nektarios Tavernarakis
Nektarios TavernarakisAffiliation 1: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Crete, Greece
Affiliation 2: Department of Basic Sciences, Faculty of Medicine, University of Crete, Crete, Greece
For correspondence: tavernarakis@imbb.forth.gr
Bio-protocol author page: a3833
date: 3/5/2017, 260 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2156.

Brief version appeared in Nature, Oct 2012
Currently available biochemical methods cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. Here, we describe a non-invasive method for monitoring protein synthesis in single cells or tissues with intrinsically different translation rates, in live Caenorhabditis elegans animals.

In Gel Kinase Assay

Featured protocol,  Authors: Gaston A. Pizzio
Gaston A. PizzioAffiliation: Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CRAG; consortium CSIC-IRTA-UAB-UB), Barcelona, Spain
For correspondence: gapizzio@gmail.com
Bio-protocol author page: a2769
 and Pedro L. Rodriguez
Pedro L. RodriguezAffiliation: Instituto de Biologia Molecular y Celular de Plantas, Consejo Superior de Investigaciones Cientificas-Universidad Politecnica de Valencia, Valencia, Spain
Bio-protocol author page: a4211
date: 3/5/2017, 233 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2170.

Brief version appeared in Plant Cell, Jun 2012
Proper spatiotemporal regulation of protein phosphorylation in cells and tissues is required for normal development and homeostasis. We present the protocol ‘In Gel Kinase Assay’, which is useful for protein kinase activity measurements from crude protein extracts. We have successfully used ‘In Gel Kinase Assay’ protocol to show that the Arabidopsis thaliana sextuple mutant in the PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYR/PYL/RCAR-ABA receptors; line pyr/pyl112458) is impaired in ABA-mediated activation of SnRK2.2, SnRK2.3 and OST1/SnRK2.6, as much as the triple mutant snrk2.2/2.3/2.6 (Gonzalez-Guzman et al., 2012).

Direct Visualization and Quantification of the Actin Nucleation and Elongation Events in vitro by TIRF Microscopy

Featured protocol,  Authors: Yuxiang Jiang
Yuxiang JiangAffiliation 1: Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing, China
Affiliation 2: Institute of Botany, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a4178
 and Shanjin Huang
Shanjin HuangAffiliation 1: Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing, China
Affiliation 2: Institute of Botany, Chinese Academy of Sciences, Beijing, China
For correspondence: sjhuang@tsinghua.edu.cn
Bio-protocol author page: a966
date: 3/5/2017, 187 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2146.

Brief version appeared in Mol Plant, Dec 2015
Total internal reflection fluorescence (TIRF) microscopy is a powerful tool for visualizing the dynamics of actin filaments at single-filament resolution in vitro. Thanks to the development of various fluorescent probes, we can easily monitor all kinds of events associated with actin dynamics, including nucleation, elongation, bundling, fragmentation and monomer dissociation. Here we present a detailed protocol regarding the visualization and quantification of actin nucleation and filament elongation events by TIRF microscopy in vitro, which is based on the methods previously reported (Liu et al., 2015; Yang et al., 2011) .

Acetyl Bromide Soluble Lignin (ABSL) Assay for Total Lignin Quantification from Plant Biomass

Featured protocol,  Authors: William J. Barnes
William J. BarnesAffiliation 1: Department of Biology, The Pennsylvania State University, University Park, PA, USA
Affiliation 2: Center for Lignocellulose Structure and Formation, The Pennsylvania State University, University Park, PA, USA
Bio-protocol author page: a4185
 and Charles T. Anderson
Charles T. AndersonAffiliation 1: Department of Biology, The Pennsylvania State University, University Park, PA, USA
Affiliation 2: Center for Lignocellulose Structure and Formation, The Pennsylvania State University, University Park, PA, USA
For correspondence: cta3@psu.edu
Bio-protocol author page: a4143
date: 3/5/2017, 216 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2149.

Brief version appeared in J Exp Bot, Jul 2015
Lignin is the second most abundant biopolymer on Earth, providing plants with mechanical support in secondary cell walls and defense against abiotic and biotic stresses. However, lignin also acts as a barrier to biomass saccharification for biofuel generation (Carroll and Somerville, 2009; Zhao and Dixon, 2011; Wang et al., 2013). For these reasons, studying the properties of lignin is of great interest to researchers in agriculture and bioenergy fields. This protocol describes the acetyl bromide method of total lignin extraction and quantification, which is favored among other methods for its high recovery, consistency, and insensitivity to different tissue types (Johnson et al., 1961; Chang et al., 2008; Moreira-Vilar et al., 2014; Kapp et al., 2015). In brief, acetyl bromide digestion causes the formation of acetyl derivatives on free hydroxyl groups and bromide substitution of α-carbon hydroxyl groups on the lignin backbone to cause a complete solubilization of lignin, which can be quantified using known extinction coefficients and absorbance at 280 nm (Moreira-Vilar et al., 2014).

Determination of Elemental Concentrations in Lichens Using ICP-AES/MS

Featured protocol,  Authors: Liang-Cheng Zhao
Liang-Cheng ZhaoAffiliation: Hebei Geological Laboratory, Baoding, China
Bio-protocol author page: a4076
Li Wang
Li WangAffiliation: Hebei Geological Laboratory, Baoding, China
Bio-protocol author page: a4077
Yun-Jun Jiang
Yun-Jun JiangAffiliation: Hebei Geological Laboratory, Baoding, China
Bio-protocol author page: a4078
Yan-Qiao Hu
Yan-Qiao HuAffiliation: Hebei Geological Laboratory, Baoding, China
Bio-protocol author page: a4122
Chong-Ying Xu
Chong-Ying XuAffiliation: Hebei Geological Laboratory, Baoding, China
Bio-protocol author page: a4080
Lei Wang
Lei WangAffiliation: Hebei Geological Laboratory, Baoding, China
Bio-protocol author page: a4081
Xing Li
Xing Li Affiliation: Hebei Geological Laboratory, Baoding, China
Bio-protocol author page: a4082
Li Wei
Li WeiAffiliation: Hebei Geological Laboratory, Baoding, China
Bio-protocol author page: a4083
Xiu-Ping Guo
Xiu-Ping GuoAffiliation: Hebei Geological Laboratory, Baoding, China
Bio-protocol author page: a4084
Ai-Qin Liu
Ai-Qin LiuAffiliation: Hebei Geological Laboratory, Baoding, China
For correspondence: laq217510@sina.com
Bio-protocol author page: a4086
 and Hua-Jie Liu
Hua-Jie LiuAffiliation: College of Life Sciences, Hebei University, Baoding, China
For correspondence: liuhuajie@foxmail.com
Bio-protocol author page: a4087
date: 3/5/2017, 161 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2165.

Brief version appeared in Sci Rep, Apr 2016
Lichens are good biomonitors for air pollution because of their high enrichment capability of atmospheric chemical elements. To monitor atmospheric element deposition using lichens, it is important to obtain information on the multi-element concentrations in lichen thalli. Because of serious air pollution, elemental concentrations in thalli of lichens from North China (especially Inner Mongolia, Hebei, Shanxi and Henan province) are often higher than those from other regions, therefore highlighting the necessity to optimize ICP-AES/MS (Inductively coupled plasma-atomic emission spectroscopy/mass spectrometry) for analyzing lichen element content. Based on the high elemental concentrations in the lichen samples, and the differences in the sensitivity and detection limits between ICP-MS and ICP-AES, we propose a protocol for analyzing 31 elements in lichens using ICP-AES/MS. Twenty-two elements (Cd, Ce, Co, Cr, Cs, Cu, K, La, Mo, Na, Ni, Pb, Rb, Sb, Sc, Sm, Sr, Tb, Th, Tl, V and Zn) can be identified by using microwave digestion- ICP-MS, and 9 elements (Al, Ba, Ca, Fe, Mg, Mn, P, S and Ti) by using ashing-alkali fusion digestion- ICP-AES.

Liposome Flotation Assays for Phosphoinositide-protein Interaction

Featured protocol,  Authors: Helene Tronchere
Helene TronchereAffiliation: INSERM U1048 I2MC and Université Paul Sabatier, Toulouse, France
Bio-protocol author page: a4209
 and Frederic Boal
Frederic BoalAffiliation: INSERM U1048 I2MC and Université Paul Sabatier, Toulouse, France
For correspondence: frederic.boal@inserm.fr
Bio-protocol author page: a4210
date: 3/5/2017, 208 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2169.

Brief version appeared in J Cell Sci, Feb 2015
Phosphoinositides are rare membrane lipids involved in the control of the major cellular functions and signaling pathways. They are able to recruit specific effector proteins to the cytosolic face of plasma membrane and organelles to coordinate a vast variety of signaling and trafficking processes, as well to maintain specific identity of the different subcellular compartments (Di Paolo and De Camilli, 2006; Lemmon, 2003). Therefore, analysis of these effectors’ binding properties and specificity towards different phosphoinositides is crucial for the understanding of their cellular functions. This protocol describes a method to characterize the binding of proteins to different phosphoinositide-containing vesicles.

Transient Transfection-based Fusion Assay for Viral Proteins

Featured protocol,  Authors: Melina Vallbracht
Melina VallbrachtAffiliation: Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany
Bio-protocol author page: a4197
Christina Schröter
Christina SchröterAffiliation: Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany
Bio-protocol author page: a4205
Barbara G. Klupp
Barbara G. KluppAffiliation: Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany
Bio-protocol author page: a4206
 and Thomas C. Mettenleiter
Thomas C. MettenleiterAffiliation: Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany
For correspondence: thomas.mettenleiter@fli.de
Bio-protocol author page: a4207
date: 3/5/2017, 178 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2162.

Brief version appeared in J Virol, Dec 2015
Membrane fusion is vital for entry of enveloped viruses into host cells as well as for direct viral cell-to-cell spread. To understand the fusion mechanism in more detail, we use an infection free system whereby fusion can be induced by a minimal set of the alphaherpesvirus pseudorabies virus (PrV) glycoproteins gB, gH and gL. Here, we describe an optimized protocol of a transient transfection based fusion assay to quantify cell-cell fusion induced by the PrV glycoproteins.

Chitin Extraction and Content Measurement in Magnaporthe oryzae

Featured protocol,  Authors: Xinyu Liu
Xinyu LiuAffiliation: Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
Bio-protocol author page: a4123
 and Zhengguang Zhang
Zhengguang ZhangAffiliation: Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
For correspondence: zhgzhang@njau.edu.cn
Bio-protocol author page: a4208
date: 3/5/2017, 202 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2164.

Brief version appeared in Mol Plant Microbe Interact, Jun 2016
Chitin is a linear polysaccharide composed of β (1→4)-linked N-acetylglucosamine (GlcNAc) residues. In fungi, chitin is an important component of the cell wall. Here, we provide a protocol to measure the chitin content of fungal cells using Magnaporthe oryzae as an example.

Isolation of Outer Membrane Vesicles from Phytopathogenic Xanthomonas campestris pv. campestris

Featured protocol,  Authors: Gideon Mordukhovich
Gideon MordukhovichAffiliation 1: Department of Plant Pathology and Weed Research, Agricultural Research Organization, Volcani Center, Rishon LeZion, Israel
Affiliation 2: Robert H. Smith Faculty of Agriculture, Food, and Environment, Hebrew University of Jerusalem, Rehovot, Israel
For correspondence: gideon.mordukhovic@mail.huji.ac.il
Bio-protocol author page: a4144
 and Ofir Bahar
Ofir BaharAffiliation: Department of Plant Pathology and Weed Research, Agricultural Research Organization, Volcani Center, Rishon LeZion, Israel
For correspondence: ofirb@agri.gov.il
Bio-protocol author page: a4145
date: 3/5/2017, 163 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2160.

Brief version appeared in Mol Plant Microbe Interact, May 2016
Gram-negative bacteria naturally release outer membrane vesicles (OMVs) to the surrounding environment. OMVs contribute to multiple processes, such as cell-cell communication, delivery of enzymes and toxins, resistance to environmental stresses and pathogenesis. Little is known about OMVs produced by plant-pathogenic bacteria, and their interactions with host plants. The protocol described below discusses the isolation process of OMVs from Xanthomonas campestris pv. campestris strain 33913, a bacterial pathogen of Crucifiers. Nevertheless, this protocol can be used and/or adapted for isolation of OMVs from other phytopathogenic bacteria to promote the study of OMVs in the context of plant-microbe interactions.

Polysome Analysis

Authors: Dipak Kumar Poria
Dipak Kumar PoriaAffiliation: Department of Biological Sciences, Indian Institute of Science Education and Research, Kolkata, India
Bio-protocol author page: a4215
 and Partho Sarothi Ray
Partho Sarothi RayAffiliation: Department of Biological Sciences, Indian Institute of Science Education and Research, Kolkata, India
For correspondence: psray@iiserkol.ac.in
Bio-protocol author page: a4216
date: 3/20/2017, 144 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2192.

[Abstract] Polysome analysis is a method to separate mRNAs from a cell into actively translating and non-translating fractions depending on their association with polysomes. By this protocol, cell lysates are fractionated by sucrose density gradient ultracentrifugation. Free mRNA fraction and various ribosomal fractions, such as 40S, 60S, monosomes and polysomes ...

Measurement of RNA-induced PKR Activation in vitro

Author: Kobe C. Yuen
Kobe C. YuenAffiliation: Stowers Institute for Medical Research, Kansas City, MO, USA
Present address: 1 DNA Way, South San Francisco, USA
For correspondence: yuenc4@gene.com
Bio-protocol author page: a4260
date: 3/20/2017, 141 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2178.

[Abstract] Protein kinase R (PKR) is one of the key RNA-activated sensors for innate immunity. PKR is activated by pathogenic or aberrant RNAs such as short double-stranded RNAs or those with imperfect secondary structures, as well as a reduction in the amount and number of RNA modifications. Activation of PKR may be an underlying mechanism for the pathogenesis ...

Chase Assay of Protein Stability in Haloferax volcanii

Authors: Xian Fu
Xian FuAffiliation: Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, USA
Bio-protocol author page: a4266
 and Julie A. Maupin-Furlow
Julie A. Maupin-FurlowAffiliation 1: Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, USA
Affiliation 2: Genetics Institute, University of Florida, Gainesville, Florida, USA
For correspondence: jmaupin@ufl.edu
Bio-protocol author page: a4267
date: 3/20/2017, 107 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2186.

[Abstract] Highly regulated and targeted protein degradation plays a fundamental role in almost all cellular processes. Determination of the protein half-life by the chase assay serves as a powerful and popular strategy to compare the protein stability and study proteolysis pathways in cells. Here, we describe a chase assay in Haloferax volcanii, a halophilic ...

Rubisco Extraction and Purification from Diatoms

Authors: Jodi N. Young
Jodi N. YoungAffiliation: Department of Oceanography, University of Washington, Seattle, USA
For correspondence: youngjn@uw.edu
Bio-protocol author page: a4026
Ana M. C. Heureux
Ana M. C. HeureuxAffiliation: Department of Earth Sciences, University of Oxford, Oxford, UK
Bio-protocol author page: a4027
Rosalind E. M. Rickaby
Rosalind E. M. RickabyAffiliation: Department of Earth Sciences, University of Oxford, Oxford, UK
Bio-protocol author page: a4028
François M. M. Morel
François M. M. MorelAffiliation: Department of Geosciences, Princeton University, Princeton, USA
Bio-protocol author page: a4029
Spencer M. Whitney
Spencer M. WhitneyAffiliation: Plant Science Division, Research School of Biology, the Australian National University, Canberra, Australia
Bio-protocol author page: a4030
 and Robert E. Sharwood
Robert E. SharwoodAffiliation: Plant Science Division, Research School of Biology, the Australian National University, Canberra, Australia
Bio-protocol author page: a4031
date: 3/20/2017, 134 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2191.

[Abstract] This protocol describes a method to extract ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) from diatoms (Bacillariophyta) to determine catalytic performance. This protocol has been adapted from use in cyanobacteria and higher plants (Andrews, 1988; Whitney and Sharwood, 2007). First part (steps A1-A3) of the extraction provides a crude extract ...

Determination of Adeno-associated Virus Rep DNA Binding Using Fluorescence Anisotropy

Authors: Francisco Zarate-Perez
Francisco Zarate-PerezAffiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond VA, USA
Bio-protocol author page: a4284
Vishaka Santosh
Vishaka SantoshAffiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond VA, USA
Bio-protocol author page: a4286
Martino Bardelli
Martino BardelliAffiliation: Department of Infectious Diseases, King’s College London, London, United Kingdom
Bio-protocol author page: a4283
Leticia Agundez
Leticia AgundezAffiliation: Department of Infectious Diseases, King’s College London, London, United Kingdom
Bio-protocol author page: a4285
R. Michael Linden
R. Michael LindenAffiliation: Department of Infectious Diseases, King’s College London, London, United Kingdom
Bio-protocol author page: a4287
Els Henckaerts
Els HenckaertsAffiliation: Department of Infectious Diseases, King’s College London, London, United Kingdom
Bio-protocol author page: a4289
 and Carlos R. Escalante
Carlos R. EscalanteAffiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond VA, USA
For correspondence: carlos.escalante@vcuhealth.org
Bio-protocol author page: a4288
date: 3/20/2017, 93 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2194.

[Abstract] Quantitative measurement of proteins binding to DNA is a requisite to fully characterize the structural determinants of complex formation necessary to understand the DNA transactions that regulate cellular processes. Here we describe a detailed protocol to measure binding affinity of the adeno-associated virus (AAV) Rep68 protein for the integration ...

Extraction, Purification and Quantification of Diffusible Signal Factor Family Quorum-sensing Signal Molecules in Xanthomonas oryzae pv. oryzae

Authors: Lian Zhou
Lian ZhouAffiliation 1: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Affiliation 2: Zhiyuan Innovative Research Center, Shanghai Jiao Tong University, Shanghai, China
Bio-protocol author page: a4225
Xing-Yu Wang
Xing-Yu WangAffiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Bio-protocol author page: a4226
Wei Zhang
Wei ZhangAffiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Bio-protocol author page: a4227
Shuang Sun
Shuang SunAffiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Bio-protocol author page: a4228
 and Ya-Wen He
Ya-Wen HeAffiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
For correspondence: yawenhe@sjtu.edu.cn
Bio-protocol author page: a4229
date: 3/20/2017, 108 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2190.

[Abstract] Bacteria use quorum-sensing (QS) systems to monitor and regulate their population density. Bacterial QS involves small molecules that act as signals for bacterial communication. Many Gram-negative bacterial pathogens use a class of widely conserved molecules, called diffusible signal factor (DSF) family QS signals. The measurement of DSF family signal ...

Isolation of Ribosomal Particles from the Unicellular Cyanobacterium Synechocystis sp. PCC 6803

Authors: Carla V. Galmozzi
Carla V. GalmozziAffiliation 1: Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla, Sevilla, Spain
Affiliation 2: Zentrum für Molekulare Biologie der Universität Heidelberg, Heidelberg, Germany
Bio-protocol author page: a4172
 and M. Isabel Muro-Pastor
M. Isabel Muro-Pastor Affiliation: Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla, Sevilla, Spain
For correspondence: imuro@ibvf.csic.es
Bio-protocol author page: a4173
date: 3/20/2017, 107 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2176.

[Abstract] Isolation of ribosomal particles is an essential step in the study of ribosomal components as well as in the analysis of trans-acting factors that interact with the ribosome to regulate protein synthesis and modulate the expression profile of the cell in response to different environmental conditions. In this protocol, we describe a procedure for the ...

Assays for the Detection of Rubber Oxygenase Activities

Authors: Wolf Röther
Wolf RötherAffiliation: Institute of Microbiology, University Stuttgart, Stuttgart, Germany
Bio-protocol author page: a4273
Jakob Birke
Jakob BirkeAffiliation: Institute of Microbiology, University Stuttgart, Stuttgart, Germany
Bio-protocol author page: a4274
 and Dieter Jendrossek
Dieter JendrossekAffiliation: Institute of Microbiology, University Stuttgart, Stuttgart, Germany
For correspondence: dieter.jendrossek@imb.uni-stuttgart.de
Bio-protocol author page: a4275
date: 3/20/2017, 117 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2188.

[Abstract] Microbial biodegradation of rubber relies on extracellular rubber oxygenases that catalyze the oxidative cleavage of the double bond of the polyisoprene backbone into oligo-isoprenoids. This protocol describes the determination of rubber oxygenase activities by an online measurement of molecular oxygen consumption via a non-invasive fluorescence-based ...

Protein Synthesis Rate Assessment by Fluorescence Recovery after Photobleaching (FRAP)

Authors: Nikos Kourtis
Nikos KourtisAffiliation 1: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Crete, Greece
Affiliation 2: Department of Pathology, NYU School of Medicine, New York, USA
Bio-protocol author page: a4141
 and Nektarios Tavernarakis
Nektarios TavernarakisAffiliation 1: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Crete, Greece
Affiliation 2: Department of Basic Sciences, Faculty of Medicine, University of Crete, Crete, Greece
For correspondence: tavernarakis@imbb.forth.gr
Bio-protocol author page: a3833
date: 3/5/2017, 260 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2156.

[Abstract] Currently available biochemical methods cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. Here, we describe a non-invasive method for monitoring protein synthesis in single cells or tissues with intrinsically different translation rates, in live Caenorhabditis elegans animals....

In Gel Kinase Assay

Authors: Gaston A. Pizzio
Gaston A. PizzioAffiliation: Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CRAG; consortium CSIC-IRTA-UAB-UB), Barcelona, Spain
For correspondence: gapizzio@gmail.com
Bio-protocol author page: a2769
 and Pedro L. Rodriguez
Pedro L. RodriguezAffiliation: Instituto de Biologia Molecular y Celular de Plantas, Consejo Superior de Investigaciones Cientificas-Universidad Politecnica de Valencia, Valencia, Spain
Bio-protocol author page: a4211
date: 3/5/2017, 233 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2170.

[Abstract] Proper spatiotemporal regulation of protein phosphorylation in cells and tissues is required for normal development and homeostasis. We present the protocol ‘In Gel Kinase Assay’, which is useful for protein kinase activity measurements from crude protein extracts. We have successfully used ‘In Gel Kinase Assay’ protocol to show that the Arabidopsis ...
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[Bio101] Bradford Protein Assay

Author: Fanglian He date: 3/20/2011, 85111 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, 63840 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, 40612 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, 40449 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, 32274 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, 31945 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, 30895 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, 29728 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, 29522 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, 24030 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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