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In vitro Histone H3 Cleavage Assay for Yeast and Chicken Liver H3 Protease

Featured protocol,  Authors: Sakshi Chauhan
Sakshi ChauhanAffiliation: Laboratory of Chromatin Biology, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, India
Bio-protocol author page: a3946
Gajendra Kumar Azad
Gajendra Kumar AzadAffiliation 1: Laboratory of Chromatin Biology, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, India
Affiliation 2: Department of Genetics, Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, Israel
Bio-protocol author page: a3947
 and Raghuvir Singh Tomar
Raghuvir Singh TomarAffiliation: Laboratory of Chromatin Biology, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, India
For correspondence: rst@iiserb.ac.in
Bio-protocol author page: a3948
date: 1/5/2017, 112 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2085.

Brief version appeared in Yeast, Jun 2016
Histone proteins are subjected to a wide array of reversible and irreversible post-translational modifications (PTMs) (Bannister and Kouzarides, 2011; Azad and Tomar, 2014). The PTMs on histones are known to regulate chromatin structure and function. Histones are irreversibly modified by proteolytic clipping of their tail domains. The proteolytic clipping of histone tails is continuously attracting interest of researchers in the field of chromatin biology. We can recapitulate H3-clipping by performing in vitro H3 cleavage assay. Here, we are presenting the detailed protocol to perform in vitro H3 cleavage assay.

Plant Tissue Trypan Blue Staining During Phytopathogen Infection

Featured protocol,  Authors: Nuria Fernández-Bautista
Nuria Fernández-BautistaAffiliation: Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) – Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Parque Científico y Tecnológico, UPM, Campus de Montegancedo, Ctra M-40, km 38, 28223, Pozuelo de Alarcón (Madrid), Spain
Bio-protocol author page: a3925
José Alfonso Domínguez-Núñez
José Alfonso Domínguez-NúñezAffiliation: Departamento de Sistemas y Recursos Naturales, Escuela Técnica Superior de Ingeniería de Montes, Forestal y del Medio Natural, Ciudad Universitaria s/n, 28040, Madrid, Spain
Bio-protocol author page: a3926
M. Mar Castellano Moreno
M. Mar Castellano MorenoAffiliation: Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) – Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Parque Científico y Tecnológico, UPM, Campus de Montegancedo, Ctra M-40, km 38, 28223, Pozuelo de Alarcón (Madrid), Spain
Bio-protocol author page: a3927
 and Marta Berrocal-Lobo
Marta Berrocal-LoboAffiliation 1: Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) – Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Parque Científico y Tecnológico, UPM, Campus de Montegancedo, Ctra M-40, km 38, 28223, Pozuelo de Alarcón (Madrid), Spain
Affiliation 2: Departamento de Sistemas y Recursos Naturales, Escuela Técnica Superior de Ingeniería de Montes, Forestal y del Medio Natural, Ciudad Universitaria s/n, 28040, Madrid, Spain
For correspondence: m.berrocal@upm.es
Bio-protocol author page: a3928
date: 12/20/2016, 214 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2078.

Brief version appeared in Plant J, Jan 2002
In this protocol plant tissue is stained with trypan blue dye allowing the researcher to visualize cell death. Specifically this method avoids the use of the carcinogen compound chloral hydrate, making this classical method of staining safer and faster than never. The protocol is applied specifically to detect cell death on Arabidopsis leaves during the course of infection with necrotrophic fungus Botrytis cinerea.

Assessment of Wheat Resistance to Fusarium graminearum by Automated Image Analysis of Detached Leaves Assay

Featured protocol,  Authors: Alexandre Perochon
Alexandre PerochonAffiliation: University College Dublin Earth Institute and School of Biology and Environmental Science, College of Science, University College Dublin, Belfield, Dublin, Ireland
For correspondence: alexandre.perochon@ucd.ie
Bio-protocol author page: a3890
 and Fiona M. Doohan
Fiona M. DoohanAffiliation: University College Dublin Earth Institute and School of Biology and Environmental Science, College of Science, University College Dublin, Belfield, Dublin, Ireland
Bio-protocol author page: a2308
date: 12/20/2016, 340 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2065.

Brief version appeared in Plant Physiol, Dec 2015
Fusarium head blight (FHB) caused by Fusarium pathogens is a globally important cereal disease. To study Fusarium pathogenicity and host disease resistance, robust methods for disease assessment and quantification are needed. Here we describe the procedure of a detached leaves assay emphasizing the image analysis. The protocol provides the different steps of a rapid, automatic and quantitative image analysis to evaluate leaf area infected by Fusarium graminearum.

Highly Accurate Real-time Measurement of Rapid Hydrogen-peroxide Dynamics in Fungi

Featured protocol,  Authors: Michael Mentges
Michael MentgesAffiliation: Biocenter Klein Flottbek, Department of Molecular Phytopathology, University of Hamburg, Hamburg, Germany
Bio-protocol author page: a3933
 and Jörg Bormann
Jörg BormannAffiliation: Biocenter Klein Flottbek, Department of Molecular Phytopathology, University of Hamburg, Hamburg, Germany
For correspondence: joerg.bormann@uni-hamburg.de
Bio-protocol author page: a3387
date: 12/20/2016, 179 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2080.

Brief version appeared in Mol Microbiol, Dec 2015
Reactive oxygen species (ROS) are unavoidable by-products of aerobic metabolism. Despite beneficial aspects as a signaling molecule, ROS are principally recognized as harmful agents that act on nucleic acids, proteins and lipids. Reactive oxygen species, and, in particular, hydrogen peroxide (H2O2), are deployed as defense molecules across kingdoms, e.g., by plants in order to defeat invading pathogens like fungi. Necrotrophic plant pathogenic fungi themselves secrete H2O2 to induce host cell death and facilitate infection. Hydrogen peroxide is, to a certain extent, freely diffusible through membranes. To be able to monitor intracellular hydrogen peroxide dynamics in fungi, we recently established the versatile HyPer-imaging technique in the filamentous plant pathogen Fusarium graminearum (Mentges and Bormann, 2015). HyPer consists of a circularly permuted yellow fluorescent protein (cpYFP) inserted into the regulatory domain (RD) of the prokaryotic H2O2-sensing protein, OxyR. The OxyR domain renders the sensor highly specific for H2O2. Oxidation of HyPer increases fluorescence of cpYFP excited at 488 nm and decreases fluorescence excited at 405 nm, thereby facilitating ratiometric readouts (Belousov et al., 2006). HyPer turned out to be pH-sensitive. A single amino acid mutation in the H2O2-sensing domain of HyPer renders the sensor insensitive to H2O2. This reporter is called SypHer and serves as a control for pH changes.

Single-step Marker Switching in Schizosaccharomyces pombe Using a Lithium Acetate Transformation Protocol

Featured protocol,  Authors: Simon David Brown
Simon David BrownAffiliation: Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK
Bio-protocol author page: a3916
 and Alexander Lorenz
Alexander LorenzAffiliation: Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK
For correspondence: a.lorenz@abdn.ac.uk
Bio-protocol author page: a3917
date: 12/20/2016, 316 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2075.

Brief version appeared in Yeast, Dec 2015
The ability to utilize different selectable markers for tagging or mutating multiple genes in Schizosaccharomyces pombe is hampered by the historical use of only two selectable markers, ura4+ and kanMX6; the latter conferring resistance to the antibiotic G418 (geneticin). More markers have been described recently, but introducing these into yeast cells often requires strain construction from scratch. To overcome this problem we and other groups have created transformation cassettes with flanking homologies to ura4+ and kanMX6 which enable an efficient and time-saving way to exchange markers in existing mutated or tagged fission yeast strains.

In vitro Histone H3 Cleavage Assay for Yeast and Chicken Liver H3 Protease

Authors: Sakshi Chauhan
Sakshi ChauhanAffiliation: Laboratory of Chromatin Biology, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, India
Bio-protocol author page: a3946
Gajendra Kumar Azad
Gajendra Kumar AzadAffiliation 1: Laboratory of Chromatin Biology, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, India
Affiliation 2: Department of Genetics, Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, Israel
Bio-protocol author page: a3947
 and Raghuvir Singh Tomar
Raghuvir Singh TomarAffiliation: Laboratory of Chromatin Biology, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, India
For correspondence: rst@iiserb.ac.in
Bio-protocol author page: a3948
date: 1/5/2017, 112 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2085.

[Abstract] Histone proteins are subjected to a wide array of reversible and irreversible post-translational modifications (PTMs) (Bannister and Kouzarides, 2011; Azad and Tomar, 2014). The PTMs on histones are known to regulate chromatin structure and function. Histones are irreversibly modified by proteolytic ...

Plant Tissue Trypan Blue Staining During Phytopathogen Infection

Authors: Nuria Fernández-Bautista
Nuria Fernández-BautistaAffiliation: Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) – Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Parque Científico y Tecnológico, UPM, Campus de Montegancedo, Ctra M-40, km 38, 28223, Pozuelo de Alarcón (Madrid), Spain
Bio-protocol author page: a3925
José Alfonso Domínguez-Núñez
José Alfonso Domínguez-NúñezAffiliation: Departamento de Sistemas y Recursos Naturales, Escuela Técnica Superior de Ingeniería de Montes, Forestal y del Medio Natural, Ciudad Universitaria s/n, 28040, Madrid, Spain
Bio-protocol author page: a3926
M. Mar Castellano Moreno
M. Mar Castellano MorenoAffiliation: Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) – Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Parque Científico y Tecnológico, UPM, Campus de Montegancedo, Ctra M-40, km 38, 28223, Pozuelo de Alarcón (Madrid), Spain
Bio-protocol author page: a3927
 and Marta Berrocal-Lobo
Marta Berrocal-LoboAffiliation 1: Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) – Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Parque Científico y Tecnológico, UPM, Campus de Montegancedo, Ctra M-40, km 38, 28223, Pozuelo de Alarcón (Madrid), Spain
Affiliation 2: Departamento de Sistemas y Recursos Naturales, Escuela Técnica Superior de Ingeniería de Montes, Forestal y del Medio Natural, Ciudad Universitaria s/n, 28040, Madrid, Spain
For correspondence: m.berrocal@upm.es
Bio-protocol author page: a3928
date: 12/20/2016, 214 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2078.

[Abstract] In this protocol plant tissue is stained with trypan blue dye allowing the researcher to visualize cell death. Specifically this method avoids the use of the carcinogen compound chloral hydrate, making this classical method of staining safer and faster than never. The protocol is applied specifically ...

Assessment of Wheat Resistance to Fusarium graminearum by Automated Image Analysis of Detached Leaves Assay

Authors: Alexandre Perochon
Alexandre PerochonAffiliation: University College Dublin Earth Institute and School of Biology and Environmental Science, College of Science, University College Dublin, Belfield, Dublin, Ireland
For correspondence: alexandre.perochon@ucd.ie
Bio-protocol author page: a3890
 and Fiona M. Doohan
Fiona M. DoohanAffiliation: University College Dublin Earth Institute and School of Biology and Environmental Science, College of Science, University College Dublin, Belfield, Dublin, Ireland
Bio-protocol author page: a2308
date: 12/20/2016, 340 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2065.

[Abstract] Fusarium head blight (FHB) caused by Fusarium pathogens is a globally important cereal disease. To study Fusarium pathogenicity and host disease resistance, robust methods for disease assessment and quantification are needed. Here we describe the procedure of a detached leaves assay emphasizing the ...

Highly Accurate Real-time Measurement of Rapid Hydrogen-peroxide Dynamics in Fungi

Authors: Michael Mentges
Michael MentgesAffiliation: Biocenter Klein Flottbek, Department of Molecular Phytopathology, University of Hamburg, Hamburg, Germany
Bio-protocol author page: a3933
 and Jörg Bormann
Jörg BormannAffiliation: Biocenter Klein Flottbek, Department of Molecular Phytopathology, University of Hamburg, Hamburg, Germany
For correspondence: joerg.bormann@uni-hamburg.de
Bio-protocol author page: a3387
date: 12/20/2016, 179 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2080.

[Abstract] Reactive oxygen species (ROS) are unavoidable by-products of aerobic metabolism. Despite beneficial aspects as a signaling molecule, ROS are principally recognized as harmful agents that act on nucleic acids, proteins and lipids. Reactive oxygen species, and, in particular, hydrogen peroxide (H2O2), ...

Single-step Marker Switching in Schizosaccharomyces pombe Using a Lithium Acetate Transformation Protocol

Authors: Simon David Brown
Simon David BrownAffiliation: Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK
Bio-protocol author page: a3916
 and Alexander Lorenz
Alexander LorenzAffiliation: Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK
For correspondence: a.lorenz@abdn.ac.uk
Bio-protocol author page: a3917
date: 12/20/2016, 316 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2075.

[Abstract] The ability to utilize different selectable markers for tagging or mutating multiple genes in Schizosaccharomyces pombe is hampered by the historical use of only two selectable markers, ura4+ and kanMX6; the latter conferring resistance to the antibiotic G418 (geneticin). More markers have been described ...

Fusarium graminearum Maize Stalk Infection Assay and Associated Microscopic Observation Protocol

Authors: Juan He
Juan HeAffiliation: National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
Bio-protocol author page: a3791
Tinglu Yuan
Tinglu Yuan Affiliation: National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
Bio-protocol author page: a3792
 and Wei-Hua Tang
Wei-Hua TangAffiliation: National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
For correspondence: whtang@sibs.ac.cn
Bio-protocol author page: a2037
date: 12/5/2016, 211 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2034.

[Abstract] The ascomycete fungus Fusarium graminearum (previously also called Gibberella zeae) causes Gibberella stalk rot in maize (Zea mays) and results in lodging and serious yield reduction. To develop methods to assess the fungal growth and symptom development in maize stalks, we present here a protocol of ...

Uptake Assay for Radiolabeled Peptides in Yeast

Authors: Melinda Hauser
Melinda HauserAffiliation: Department of Microbiology, University of Tennessee, Knoxville, USA
Bio-protocol author page: a3756
Houjian Cai
Houjian CaiAffiliation: Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, USA
Bio-protocol author page: a3755
Fred Naider
Fred NaiderAffiliation 1: Department of Chemistry and Macromolecular Assembly Institute, College of Staten Island of the City University of New York, Staten Island, New York, USA
Affiliation 2: Ph.D. Programs in Biochemistry and Chemistry, The Graduate Center of the City University of New York, New York, USA
Bio-protocol author page: a3757
 and Jeffrey M. Becker
Jeffrey M. BeckerAffiliation: Department of Microbiology, University of Tennessee, Knoxville, USA
For correspondence: jbecker@utk.edu
Bio-protocol author page: a3758
date: 11/20/2016, 254 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2026.

[Abstract] We describe an assay for measuring the uptake of radioactive peptides into the yeast Saccharomyces cerevisiae. The methods presented here can be adapted to measure a variety of substrates transported into any bacterial or fungal cell via specific carrier-mediated systems....

Halo Assay for Toxic Peptides and Other Compounds in Microorganisms

Authors: Houjian Cai
Houjian CaiAffiliation: Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, USA
Bio-protocol author page: a3755
Melinda Hauser
Melinda HauserAffiliation: Department of Microbiology, University of Tennessee, Knoxville, USA
Bio-protocol author page: a3756
Fred Naider
Fred NaiderAffiliation 1: Department of Chemistry and Macromolecular Assembly Institute, College of Staten Island of the City University of New York, Staten Island, USA
Affiliation 2: Ph.D. Programs in Biochemistry and Chemistry, The Graduate Center of the City University of New York, New York, USA
Bio-protocol author page: a3757
 and Jeffrey M. Becker
Jeffrey M. BeckerAffiliation: Department of Microbiology, University of Tennessee, Knoxville, USA
For correspondence: jbecker@utk.edu
Bio-protocol author page: a3758
date: 11/20/2016, 272 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2025.

[Abstract] We describe an assay for determination of toxicity in S. cerevisiae involving spotting of a toxic peptide on a lawn of yeast cells. This assay may be generalized to determine toxicity of a variety of compounds by substituting a putative toxic compound in place of the peptide. The general protocol may ...

An Assay to Study Botrytis cinerea-infected Grapevine Leaves Primed with Pseudomonas fluorescens

Authors: Charlotte Gruau
Charlotte GruauAffiliation: URVVC EA4707, UFR Sciences, University of Reims, Reims, France
Bio-protocol author page: a3539
Patricia Trotel-Aziz
Patricia Trotel-AzizAffiliation: URVVC EA4707, UFR Sciences, University of Reims, Reims, France
Bio-protocol author page: a3540
Bas Verhagen
Bas VerhagenAffiliation: URVVC EA4707, UFR Sciences, University of Reims, Reims, France
Bio-protocol author page: a3541
Sandra Villaume
Sandra VillaumeAffiliation: URVVC EA4707, UFR Sciences, University of Reims, Reims, France
Bio-protocol author page: a3542
Fanja Rabenoelina
Fanja RabenoelinaAffiliation: URVVC EA4707, UFR Sciences, University of Reims, Reims, France
Bio-protocol author page: a3543
Barbara Courteaux
Barbara CourteauxAffiliation: URVVC EA4707, UFR Sciences, University of Reims, Reims, France
Bio-protocol author page: a3544
Christophe Clément
Christophe ClémentAffiliation: URVVC EA4707, UFR Sciences, University of Reims, Reims, France
Bio-protocol author page: a3545
Fabienne Baillieul
Fabienne BaillieulAffiliation: URVVC EA4707, UFR Sciences, University of Reims, Reims, France
Bio-protocol author page: a3546
 and Aziz Aziz
Aziz AzizAffiliation: URVVC EA4707, UFR Sciences, University of Reims, Reims, France
For correspondence: aziz.aziz@univ-reims.fr
Bio-protocol author page: a3547
date: 10/5/2016, 455 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1943.

[Abstract] Grapevine (Vitis vinifera L.) is susceptible to an array of diseases among them the grey mold caused by the necrotrophic fungus Botrytis cinerea that decreases grape productivity and quality. To ensure a satisfactory yield and harvest quality numerous chemical fungicides are required, but they have ...

Flow Cytometry of Lung and Bronchoalveolar Lavage Fluid Cells from Mice Challenged with Fluorescent Aspergillus Reporter (FLARE) Conidia

Authors: Anupam Jhingran
Anupam JhingranAffiliation: Infectious Disease Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, USA
Bio-protocol author page: a3498
Shinji Kasahara
Shinji KasaharaAffiliation: Infectious Disease Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, USA
Bio-protocol author page: a3499
 and Tobias M Hohl
Tobias M HohlAffiliation 1: Infectious Disease Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, USA
Affiliation 2: Immunology Program, Sloan Kettering Institute; Memorial Sloan Kettering Cancer Center, New York, USA
For correspondence: hohlt@mskcc.org
Bio-protocol author page: a3500
date: 9/20/2016, 482 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1927.

[Abstract] Aspergillus fumigatus is a ubiquitous fungal pathogen that forms airborne conidia. The process of restricting conidial germination into hyphae by lung leukocytes is critical in determining infectious outcomes. Tracking the outcome of conidia-host cell encounters in vivo is technically challenging and ...
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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, 20654 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, 19906 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....

[Bio101] Protocol for Whole Cell Lysis of 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: 1/5/2011, 17520 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.14.

[Abstract] This protocol describes how to perform lysis on whole yeast cell samples using NaOH. The lysed cells can then be used for downstream applications such as the extraction of total proteins. ...

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, 17059 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] 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, 13761 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] How to Use an Avestin Emulsiflex C3 Homogenizer to Disrupt Cells

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, 10900 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.11.

[Abstract] The EmulsiFlex-C3 homogenizer is powered by an electric motor. The pump does not require a compressor for it to run. This equipment can be used to disrupt cells at a large scale. The EmulsiFlex-C3 has a fixed flow-through capacity of 3 L/h. It has the ability to process samples as small as 10 ml. The ...

[Bio101] Large Scale Native Affinity Purifications of Solubilized Membrane Proteins from Yeast

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicin, Baltimore , USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
date: 1/5/2011, 10583 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.12.

[Abstract] This protocol can be used to purify membrane proteins from yeast samples under native conditions at a large scale. This protocol has been developed primarily for FLAG-tagged proteins. This protocol can however be slightly modified and applied to other tags, such as GST or HA....

[Bio101] Small Scale Native Affinity Purifications of Solubilized Membrane Proteins from 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: 1/5/2011, 10039 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.15.

[Abstract] In this protocol, we show how to purify membrane proteins from yeast using affinity purification under native conditions at a small scale. ...

Illumina Sequencing Library Construction from ChIP DNA

Author: Wei Zheng
Wei ZhengAffiliation: Keck Biotech Services, Yale University, New Haven, USA
For correspondence: wei.zheng.madison@gmail.com
Bio-protocol author page: a10
date: 2/20/2012, 9416 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.91.

[Abstract] The Illumina sequencing platform is very popular among next-generation sequencing platforms. However, the DNA sequencing library construction kit provided by Illumina is considerably expensive. The protocol described here can be used to construct high-quality sequencing libraries from chromatin immunoprecipitated ...

Co-immunoprecipitation in Yeast

Author: Olesya O. Panasenko
Olesya O. PanasenkoAffiliation: Department of Microbiology and Molecular Medicine, University of Geneva, Faculty of Medicine, CMU, Geneva, Switzerland
For correspondence: olesya.panasenko@unige.ch
Bio-protocol author page: a88
date: 8/20/2012, 8867 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.250.

[Abstract] This protocol describes investigation of protein-protein interactions in baker yeast by co-immunoprecipitation (CoIP). CoIP is a technique to identify physiologically relevant protein-protein interactions in the cell. The interesting protein can be isolated out of solution using antibody that specifically ...
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