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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.

Determination of the Predatory Capability of Bdellovibrio bacteriovorus HD100

Featured protocol,  Authors: Cristina Herencias
Cristina HerenciasAffiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain
Bio-protocol author page: a4258
M. Auxiliadora Prieto
M. Auxiliadora PrietoAffiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain
Bio-protocol author page: a4259
 and Virginia Martínez
Virginia MartínezAffiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain
Present address: Evolva Biotech A/S, Copenhagen, Denmark
For correspondence: virginiaml83@gmail.com
Bio-protocol author page: a4257
date: 3/20/2017, 116 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2177.

Brief version appeared in Sci Rep, Apr 2016
Bdellovibrio bacteriovorus HD100 is an obligate predator that preys upon a wide variety of Gram negative bacteria. The biphasic growth cycle of Bdellovibrio includes a free-swimming attack phase and an intraperiplasmic growth phase, where the predator replicates its DNA and grows using the prey as a source of nutrients, finally dividing into individual cells (Sockett, 2009). Due to its obligatory predatory lifestyle, manipulation of Bdellovibrio requires two-member culturing techniques using selected prey microorganisms (Lambert et al., 2003). In this protocol, we describe a detailed workflow to grow and quantify B. bacteriovorus HD100 and its predatory ability, to easily carry out these laborious and time-consuming techniques.

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.

In vitro Assay to Assess Efficacy of Potential Antiviral Compounds against Enterovirus D68

Featured protocol,  Authors: Liang Sun
Liang SunAffiliation: KU Leuven – University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium
Bio-protocol author page: a4263
Leen Delang
Leen DelangAffiliation: KU Leuven – University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium
Bio-protocol author page: a4264
Carmen Mirabelli
Carmen Mirabelli Affiliation: KU Leuven – University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium
Bio-protocol author page: a4265
 and Johan Neyts
Johan NeytsAffiliation: KU Leuven – University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium
For correspondence: johan.neyts@kuleuven.be
Bio-protocol author page: a1546
date: 3/20/2017, 85 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2183.

Brief version appeared in Antimicrob Agents Chemother, Dec 2015
In 2014 enterovirus D68 (EV-D68) caused the largest outbreak in the United States since the discovery of the virus. Distinct from before, the 2014 infections were associated with more severe respiratory disease and occasional neurological complications. So far, there are no available vaccines or antivirals for the prophylaxis or treatment of EV-D68 infections. In order to evaluate the antiviral activity of potential inhibitors of EV-D68 replication, a cell-based cytopathic effect (CPE) reduction assay was developed (Sun et al., 2015).

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.

Wheat Root-dip Inoculation with Fusarium graminearum and Assessment of Root Rot Disease Severity

Featured protocol,  Authors: Qing Wang
Qing Wang Affiliation: Department of Plant Breeding, IFZ Research Centre for Biosystems, Land Use and Nutrition, Justus Liebig University, Giessen, Germany
Bio-protocol author page: a4241
 and Sven Gottwald
Sven GottwaldAffiliation: Department of Plant Breeding, IFZ Research Centre for Biosystems, Land Use and Nutrition, Justus Liebig University, Giessen, Germany
For correspondence: sv.gottwald@t-online.de
Bio-protocol author page: a4240
date: 3/20/2017, 127 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2189.

Brief version appeared in Mol Plant Microbe Interact, Dec 2015
Fusarium graminearum is one of the most common and potent fungal pathogens of wheat (Triticum aestivum) and other cereals, known for causing devastating yield losses and mycotoxin contaminations of food and feed. The pathogen is mainly considered as a paradigm for the floral disease Fusarium head blight, while its ability to colonize wheat plants via root infection has been examined recently. F. graminearum has a unique infection strategy which comprises complex, specialized structures and processes. Root colonisation negatively affects plant development and leads to systemic plant invasion by tissue-adapted fungal strategies. The pathosystem wheat root - F. graminearum makes available an array of research areas, such as (i) the relatively unknown root interactions with a necrotrophic pathogen; (ii) genes and pathways contributing to (overall) Fusarium resistance; (iii) induced systemic (whole-plant) resistance; (iv) pathogenic strategies in a variety of host tissues; and (v) age-related changes in the single-genotype responses to seedling and adult plant (root/spike) infection. The presented Fusarium root rot bioassay allows for efficient infection of wheat roots, evaluation of disease severity and progress as well as statistical analysis of disease dynamics.

Xylem Sap Extraction Method from Hop Plants

Featured protocol,  Authors: Marko Flajšman*
Marko FlajšmanAffiliation: Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
Bio-protocol author page: a4167
Stanislav Mandelc*
Stanislav MandelcAffiliation: Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
Bio-protocol author page: a4168
Sebastjan Radišek*
Sebastjan RadišekAffiliation: Slovenian Institute of Hop Research and Brewing, Zalec, Slovenia
Bio-protocol author page: a4169
 and Branka Javornik
Branka JavornikAffiliation: Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
For correspondence: branka.javornik@bf.uni-lj.si
Bio-protocol author page: a4170
 (*contributed equally to this work) date: 3/20/2017, 121 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2172.

Brief version appeared in Mol Plant Microbe Interact, May 2016
Verticillium wilt is one of the most important diseases on hop that significantly influence continuation of production on affected areas. It is caused by the soil borne vascular pathogen Verticillium nonalfalfae, which infects plants through the roots and then advances through the vascular (xylem) system. During infection, V. nonalfalfae secretes many different virulence factors. Xylem sap of infected plants is therefore a rich source for investigating the molecules that are involved in molecular interactions of Verticillium – hop plants. This protocol provides instructions on how to infect hop plants with V. nonalfalfae artificially and how to obtain xylem sap from hop plants.

Pathogenicity Assay of Verticillium nonalfalfae on Hop Plants

Featured protocol,  Authors: Marko Flajšman*
Marko FlajšmanAffiliation: Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
Bio-protocol author page: a4167
Sebastjan Radišek*
Sebastjan RadišekAffiliation: Slovenian Institute of Hop Research and Brewing, Zalec, Slovenia
Bio-protocol author page: a4169
 and Branka Javornik
Branka JavornikAffiliation: Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
For correspondence: branka.javornik@bf.uni-lj.si
Bio-protocol author page: a4170
 (*contributed equally to this work) date: 3/20/2017, 106 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2171.

Brief version appeared in Mol Plant Microbe Interact, May 2016
Verticillium nonalfalfae is a soil-borne plant pathogen that infects its hosts through roots. It spreads in the plant’s xylem and causes wilt disease symptoms by secreting different virulence factors. Hop (Humulus lupulus) is a primary host of V. nonalfalfae, so it is used as a model plant for studying this phytopathogenic fungus. Artificial infections of hop plants and disease scoring are prerequisites for studying the pathogen’s virulence/pathogenicity and its interaction with hop plants. In this protocol we describe the root dipping inoculation method for conducting pathogenicity assay of V. nonalfalfae on hop plants.

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).

Next-generation Sequencing of the DNA Virome from Fecal Samples

Featured protocol,  Authors: Cynthia L. Monaco
Cynthia L. MonacoAffiliation: Department of Medicine, Washington University School of Medicine, St. Louis, USA
For correspondence: cmonaco@wustl.edu
Bio-protocol author page: a4038
 and Douglas S. Kwon
Douglas S. KwonAffiliation 1: Ragon Institute of MGH, MIT, and Harvard, Cambridge, USA
Affiliation 2: Division of Infectious Diseases, Massachusetts General Hospital and Harvard Medical School, Boston, USA
Bio-protocol author page: a4039
date: 3/5/2017, 199 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2159.

Brief version appeared in Cell Host Microbe, Mar 2016
Herein we describe a detailed protocol for DNA virome analysis of low input human stool samples (Monaco et al., 2016). This protocol is divided into four main steps: 1) stool samples are pulverized to evenly distribute microbial matter; 2) stool is enriched for virus-like particles and DNA is extracted by phenol-chloroform; 3) purified DNA is multiple-strand displacement amplified (MDA) and fragmented; and 4) libraries are constructed and sequenced using Illumina Miseq. Subsequent sequence analysis for viral sequence identification should be sensitive but stringent.

Polyethylene Glycol-mediated Transformation of Drechmeria coniospora

Featured protocol,  Authors: Le D. He
Le D. HeAffiliation: Aix Marseille Univ, CNRS, INSERM, CIML, Centre d’Immunologie de Marseille-Luminy, Marseille, France
Bio-protocol author page: a4195
 and Jonathan J. Ewbank
Jonathan J. EwbankAffiliation: Aix Marseille Univ, CNRS, INSERM, CIML, Centre d’Immunologie de Marseille-Luminy, Marseille, France
For correspondence: ewbank@ciml.univ-mrs.fr
Bio-protocol author page: a4196
date: 3/5/2017, 193 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2157.

Brief version appeared in PLoS Genet, May 2016
Drechmeria coniospora is a nematophagous fungus and potential biocontrol agent. It belongs to the Ascomycota. It is related to Hirsutella minnesotensis, another nematophagous fungus but, phylogenetically, it is currently closest to the truffle parasite Tolypocladium ophioglossoides. Together with its natural host, Caenorhabditis elegans, it is used to study host-pathogen interactions. Here, we report a polyethylene glycol-mediated transformation method (Turgeon et al., 2010; Ochman et al., 1988) for this fungus. The protocol can be used to generate both knock-in or knock-out strains (Lebrigand et al., 2016).

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.

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.

Surface Inoculation and Quantification of Pseudomonas syringae Population in the Arabidopsis Leaf Apoplast

Featured protocol,  Authors: Cristián Jacob*
Cristián JacobAffiliation: Department of Plant Sciences, University of California, Davis, USA
For correspondence: cjjacob@ucdavis.edu
Bio-protocol author page: a4204
Shweta Panchal*
Shweta PanchalAffiliation: Centre for Genome Research, Faculty of Science, the Maharaja Sayajirao University of Baroda, Baroda, India
For correspondence: shwetapanchal84@gmail.com
Bio-protocol author page: a4158
 and Maeli Melotto
Maeli MelottoAffiliation: Department of Plant Sciences, University of California, Davis, USA
For correspondence: melotto@ucdavis.edu
Bio-protocol author page: a4160
 (*contributed equally to this work) date: 3/5/2017, 239 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2167.

Brief version appeared in Front Plant Sci, Jun 2016
Bacterial pathogens must enter the plant tissue in order to cause a successful infection. Foliar bacterial pathogens that are not able to directly penetrate the plant epidermis rely on wounds or natural openings to internalize leaves. This protocol describes a procedure to estimate the population size of Pseudomonas syringae in the leaf apoplast after surface inoculation of Arabidopsis rosettes.

An HPLC-based Method to Quantify Coronatine Production by Bacteria

Featured protocol,  Authors: Shweta Panchal
Shweta PanchalAffiliation: Centre for Genome Research, Department of Microbiology and Biotechnology Centre, the Maharaja Sayajirao University of Baroda, Baroda, India
For correspondence: shwetapanchal84@gmail.com
Bio-protocol author page: a4158
Zachary S. Breitbach
Zachary S. BreitbachAffiliation: Department of Chemistry, University of Texas, Arlington, USA
For correspondence: zachbreitbach@yahoo.com
Bio-protocol author page: a4159
 and Maeli Melotto
Maeli MelottoAffiliation: Department of Plant Sciences, University of California, Davis, USA
For correspondence: melotto@ucdavis.edu
Bio-protocol author page: a4160
date: 3/5/2017, 232 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2147.

Brief version appeared in Front Plant Sci, Jun 2016
Coronatine is a polyketide phytotoxin produced by several pathovars of the plant pathogenic bacterium Pseudomonas syringae. It is one of the most important virulence factors determining the success of bacterial pathogenesis in the plant at both epiphytic and endophytic stages of the disease cycle. This protocol describes an optimized procedure to culture bacterial cells for coronatine production and to quantify the amount of coronatine secreted in the culture medium using an HPLC-based method.

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.

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 ...

Determination of the Predatory Capability of Bdellovibrio bacteriovorus HD100

Authors: Cristina Herencias
Cristina HerenciasAffiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain
Bio-protocol author page: a4258
M. Auxiliadora Prieto
M. Auxiliadora PrietoAffiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain
Bio-protocol author page: a4259
 and Virginia Martínez
Virginia MartínezAffiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain
Present address: Evolva Biotech A/S, Copenhagen, Denmark
For correspondence: virginiaml83@gmail.com
Bio-protocol author page: a4257
date: 3/20/2017, 116 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2177.

[Abstract] Bdellovibrio bacteriovorus HD100 is an obligate predator that preys upon a wide variety of Gram negative bacteria. The biphasic growth cycle of Bdellovibrio includes a free-swimming attack phase and an intraperiplasmic growth phase, where the predator replicates its DNA and grows using the prey as a source of nutrients, finally dividing into individual ...

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 ...

In vitro Assay to Assess Efficacy of Potential Antiviral Compounds against Enterovirus D68

Authors: Liang Sun
Liang SunAffiliation: KU Leuven – University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium
Bio-protocol author page: a4263
Leen Delang
Leen DelangAffiliation: KU Leuven – University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium
Bio-protocol author page: a4264
Carmen Mirabelli
Carmen Mirabelli Affiliation: KU Leuven – University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium
Bio-protocol author page: a4265
 and Johan Neyts
Johan NeytsAffiliation: KU Leuven – University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium
For correspondence: johan.neyts@kuleuven.be
Bio-protocol author page: a1546
date: 3/20/2017, 85 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2183.

[Abstract] In 2014 enterovirus D68 (EV-D68) caused the largest outbreak in the United States since the discovery of the virus. Distinct from before, the 2014 infections were associated with more severe respiratory disease and occasional neurological complications. So far, there are no available vaccines or antivirals for the prophylaxis or treatment of EV-D68 ...

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 ...

Wheat Root-dip Inoculation with Fusarium graminearum and Assessment of Root Rot Disease Severity

Authors: Qing Wang
Qing Wang Affiliation: Department of Plant Breeding, IFZ Research Centre for Biosystems, Land Use and Nutrition, Justus Liebig University, Giessen, Germany
Bio-protocol author page: a4241
 and Sven Gottwald
Sven GottwaldAffiliation: Department of Plant Breeding, IFZ Research Centre for Biosystems, Land Use and Nutrition, Justus Liebig University, Giessen, Germany
For correspondence: sv.gottwald@t-online.de
Bio-protocol author page: a4240
date: 3/20/2017, 127 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2189.

[Abstract] Fusarium graminearum is one of the most common and potent fungal pathogens of wheat (Triticum aestivum) and other cereals, known for causing devastating yield losses and mycotoxin contaminations of food and feed. The pathogen is mainly considered as a paradigm for the floral disease Fusarium head blight, while its ability to colonize wheat plants via ...

Xylem Sap Extraction Method from Hop Plants

Authors: Marko Flajšman*
Marko FlajšmanAffiliation: Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
Bio-protocol author page: a4167
Stanislav Mandelc*
Stanislav MandelcAffiliation: Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
Bio-protocol author page: a4168
Sebastjan Radišek*
Sebastjan RadišekAffiliation: Slovenian Institute of Hop Research and Brewing, Zalec, Slovenia
Bio-protocol author page: a4169
 and Branka Javornik
Branka JavornikAffiliation: Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
For correspondence: branka.javornik@bf.uni-lj.si
Bio-protocol author page: a4170
 (*contributed equally to this work) date: 3/20/2017, 121 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2172.

[Abstract] Verticillium wilt is one of the most important diseases on hop that significantly influence continuation of production on affected areas. It is caused by the soil borne vascular pathogen Verticillium nonalfalfae, which infects plants through the roots and then advances through the vascular (xylem) system. During infection, V. nonalfalfae secretes many ...

Pathogenicity Assay of Verticillium nonalfalfae on Hop Plants

Authors: Marko Flajšman*
Marko FlajšmanAffiliation: Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
Bio-protocol author page: a4167
Sebastjan Radišek*
Sebastjan RadišekAffiliation: Slovenian Institute of Hop Research and Brewing, Zalec, Slovenia
Bio-protocol author page: a4169
 and Branka Javornik
Branka JavornikAffiliation: Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
For correspondence: branka.javornik@bf.uni-lj.si
Bio-protocol author page: a4170
 (*contributed equally to this work) date: 3/20/2017, 106 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2171.

[Abstract] Verticillium nonalfalfae is a soil-borne plant pathogen that infects its hosts through roots. It spreads in the plant’s xylem and causes wilt disease symptoms by secreting different virulence factors. Hop (Humulus lupulus) is a primary host of V. nonalfalfae, so it is used as a model plant for studying this phytopathogenic fungus. Artificial infections ...

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 ...
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[Bio101] Plasmid DNA Extraction from E. coli Using Alkaline Lysis Method

Author: Fanglian He
Fanglian HeAffiliation: Department of Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: fanglian09@gmail.com
Bio-protocol author page: a9
date: 2/5/2011, 85875 views, 31 Q&A
DOI: https://doi.org/10.21769/BioProtoc.30.

[Abstract] This is a quick and efficient way to extract E. coli plasmid DNA without using commercial kits....

[Bio101] E. coli Genomic DNA Extraction Updates
The author made some updates (highlighted in blue) to the protocol on 09/12/2016.

Author: Fanglian He
Fanglian HeAffiliation: Department of Biology, University of Pennsylvania, Philadelphia, USA
Bio-protocol author page: a9
date: 7/20/2011, 78981 views, 46 Q&A
DOI: https://doi.org/10.21769/BioProtoc.97.

[Abstract] This protocol uses phenol/chloroform method to purify genomic DNA without using commercial kits....

[Bio101] Lentivirus Production

Author: Nabila Aboulaich date: 3/5/2011, 22841 views, 6 Q&A
DOI: https://doi.org/10.21769/BioProtoc.39.

[Abstract] Lentivirus is a common tool used to introduce a gene into mammalian or other animal cells.This protocol is to produce lentivirus stocks from hairpin-pLKO.1 plasmid....

In vitro Protein Kinase Assay

Author: Yuehua Wei
Yuehua WeiAffiliation: Department of Pharmacology, Cancer Institute of New Jersey, UMDNJ Robert Wood Johnson Medical School, Piscataway, USA
For correspondence: weiyh.sjtu.edu@gmail.com
Bio-protocol author page: a49
date: 6/5/2012, 21457 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.212.

[Abstract] This protocol will describe experimental procedures for an in vitro kinase assay of the yeast protein kinase Sch9. This protocol can be tailored to detect kinase activity of other yeast protein kinase....

[Bio101] Making Yeast Competent Cells and Yeast Cell Transformation

Author: Yongxian Lu
Yongxian LuAffiliation: Carnegie Institution for Science, Stanford University, Stanford, USA
For correspondence: yxlu@stanford.edu
Bio-protocol author page: a28
date: 7/20/2011, 20797 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.96.

[Abstract] This is a quite simple but reliable protocol to make very high transformation efficiency yeast competent cells. By express your gene of interest, protein function can be studied in yeast cells....

Spot Assay for Yeast

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
date: 1/5/2012, 17991 views, 3 Q&A
DOI: https://doi.org/10.21769/BioProtoc.16.

[Abstract] This protocol can be used to compare the cell growth rate of yeast under different growth conditions. It involves the serial dilution and spotting of yeast colonies....

[Bio101] Purification of Adenovirus by Cesium Chloride Density Gradients

Author: Huan Pang
Huan PangAffiliation: Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, USA
For correspondence: pang_huan@hotmail.com
Bio-protocol author page: a48
date: 4/5/2012, 17507 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.201.

[Abstract] Adenovirus are efficient gene delivery systems. The standard method for purification of adenoviral vectors is based on using a cesium chloride (CsCl) density gradient combined with ultracentrifugation. This method is suitable for small-scale purification and is less expensive than column chromatography ...

Culture and Detection of Mycobacterium tuberculosis (MTB) and Mycobacterium bovis (BCG)

Author: Ran Chen
Ran ChenAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: rcchen@jfkbio.com
Bio-protocol author page: a34
date: 1/20/2012, 16881 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.49.

[Abstract] Mycobacterium tuberculosis (MTB) is the bacterial pathogen responsible for tuberculosis, a human pulmonary infectious disease. Mycobacterium bovis (BCG) is the causative agent of tuberculosis in cattle, and is often used as the vaccine stain in humans. Specific recipes and methods for culture of MTB ...

[Bio101] Yeast Vacuole Staining with FM4-64

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
date: 1/5/2011, 14336 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.18.

[Abstract] The lipophilic probe, FM 4-64 does not fluoresce much in water but fluoresces strongly after binding to the outer plasma membrane, providing clear and distinguishable plasma membrane staining. The binding is rapid and reversible. In this protocol vacuoles in yeast cells are stained with the FM4-64 dye, ...

[Bio101] Purification of 6x His-tagged Protein (from E. coli)

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
date: 1/5/2011, 13153 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.8.

[Abstract] A polyhistidine-tag is an amino acid motif that contains at least six histidine (His) residues, usually at the N- or C-terminus of the protein. This tag can also be referred to as a hexa histidine-tag or a 6x His-tag. The protocol described here has been developed to purify His-tagged proteins from ...
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