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Bacterial Intracellular Sodium Ion Measurement using CoroNa Green

Featured protocol,  Authors: Yusuke V. Morimoto
Yusuke V. MorimotoAffiliation 1: Quantitative Biology Center, RIKEN, Suita, Osaka, Japan
Affiliation 2: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan
Bio-protocol author page: a3969
Keiichi Namba
Keiichi NambaAffiliation 1: Quantitative Biology Center, RIKEN, Suita, Osaka, Japan
Affiliation 2: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan
Bio-protocol author page: a3970
 and Tohru Minamino
Tohru MinaminoAffiliation: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan
For correspondence: tohru@fbs.osaka-u.ac.jp
Bio-protocol author page: a3971
date: 1/5/2017, 109 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2092.

Brief version appeared in PLoS Pathog, Mar 2016
The bacterial flagellar type III export apparatus consists of a cytoplasmic ATPase complex and a transmembrane export gate complex, which are powered by ATP and proton motive force (PMF) across the cytoplasmic membrane, respectively, and transports flagellar component proteins from the cytoplasm to the distal end of the growing flagellar structure where their assembly occurs (Minamino, 2014). The export gate complex can utilize sodium motive force in addition to PMF when the cytoplasmic ATPase complex does not work properly. A transmembrane export gate protein FlhA acts as a dual ion channel to conduct both H+ and Na+ (Minamino et al., 2016). Here, we describe how to measure the intracellular Na+ concentrations in living Escherichia coli cells using a sodium-sensitive fluorescent dye, CoroNa Green (Minamino et al., 2016). Fluorescence intensity measurements of CoroNa Green by epi-fluorescence microscopy allows us to measure the intracellular Na+ concentration quantitatively.

Measuring Oxidative Stress in Caenorhabditis elegans: Paraquat and Juglone Sensitivity Assays

Featured protocol,  Authors: Megan M. Senchuk
Megan M. SenchukAffiliation: Laboratory of Aging and Neurodegenerative Disease (LAND), Center for Neurodegenerative Science, Van Andel Research Institute, Grand Rapids, USA
Bio-protocol author page: a3949
Dylan J. Dues
Dylan J. Dues Affiliation: Laboratory of Aging and Neurodegenerative Disease (LAND), Center for Neurodegenerative Science, Van Andel Research Institute, Grand Rapids, USA
Bio-protocol author page: a3950
 and Jeremy M. Van Raamsdonk
Jeremy M. Van RaamsdonkAffiliation: Laboratory of Aging and Neurodegenerative Disease (LAND), Center for Neurodegenerative Science, Van Andel Research Institute, Grand Rapids, USA
For correspondence: jeremy.vanraamsdonk@vai.org
Bio-protocol author page: a3951
date: 1/5/2017, 106 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2086.

Brief version appeared in PLoS Genet, Feb 2016
Oxidative stress has been proposed to be one of the main causes of aging and has been implicated in the pathogenesis of many diseases. Sensitivity to oxidative stress can be measured by quantifying survival following exposure to a reactive oxygen species (ROS)-generating compound such as paraquat or juglone. Sensitivity to oxidative stress is a balance between basal levels of ROS, the ability to detoxify ROS, and the ability to repair ROS-mediated damage.

Cation (Ca2+ and Mn2+) Partitioning Assays with Intact Arabidopsis Chloroplasts

Featured protocol,  Authors: Anna Harms
Anna HarmsAffiliation: Biozentrum der LMU München, Department Biologie I, Munich, Germany
Bio-protocol author page: a3963
Iris Steinberger
Iris SteinbergerAffiliation: Biozentrum der LMU München, Department Biologie I, Munich, Germany
Bio-protocol author page: a2369
 and Anja Schneider
Anja SchneiderAffiliation: Biozentrum der LMU München, Department Biologie I, Munich, Germany
For correspondence: anja.schneider@lrz.uni-muenchen.de
Bio-protocol author page: a2371
date: 1/5/2017, 90 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2094.

Brief version appeared in Plant J, Apr 2014
Determination of the relative distribution of Ca2+ and Mn2+ is an important tool for analyzing mutants showing altered levels of calcium and/or manganese transporters in the chloroplast envelope or thylakoid membrane. The method described in this protocol allows quantitative analyses of the relative distribution of calcium and manganese ions between chloroplast stroma and thylakoids using the isotopes [45Ca] and [54Mn] as radioactive tracers. To avoid contaminations with non chloroplastidic membrane systems, the method is designed for isolating pure and intact chloroplasts of Arabidopsis thaliana. Intact chloroplasts are isolated via Percoll gradient centrifugation. Chloroplasts are then allowed to take up [45Ca] or [54Mn] during a light incubation step. After incubation, chloroplasts are either kept intact or osmotically/mechanically treated to release thylakoids. The amount of incorporated [45Ca] or [54Mn] can be determined by liquid scintillation counting and the relative distribution calculated.

In vitro Ubiquitin Dimer Formation Assay

Featured protocol,  Authors: Sheng Wang
Sheng WangAffiliation: Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada
Bio-protocol author page: a1483
Ling Cao
Ling CaoAffiliation 1: Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada
Affiliation 2: National Key laboratory of Crop Genetic Improvement, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China
Bio-protocol author page: a1482
 and Hong Wang
Hong WangAffiliation: Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada
For correspondence: hong.wang@usask.ca
Bio-protocol author page: a1485
date: 1/5/2017, 108 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2082.

Brief version appeared in J Exp Bot, May 2016
The process of protein ubiquitination typically consists of three sequential steps to add an ubiquitin (Ub) or Ub chain to a substrate protein, requiring three different enzymes, ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin protein ligase (E3). Most E2s possess the classical E2 activity in forming E2-Ub complex through a thioester linkage, in presence of an E1 and Ub. Additionally, some E2s have the ability of catalyzing the formation of free Ub dimer. Such activity indicates an important role of these E2s in ubiquitination pathway. Thus, we developed an in vitro Ub dimer formation assay to determine the activity of certain E2s. Moreover, by using Ub mutants, in which different lysine residues are mutated, the specific linkage of dimer can also be determined.

Examination of the Interaction between a Membrane Active Peptide and Artificial Bilayers by Dual Polarisation Interferometry

Featured protocol,  Authors: Jennifer A.E. Payne
Jennifer A.E. PayneAffiliation 1: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia
Affiliation 2: Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia
For correspondence: jennifer.payne@monash.edu
Bio-protocol author page: a3952
Tzong-Hsien Lee
Tzong-Hsien LeeAffiliation: Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia
Bio-protocol author page: a3953
Marilyn A. Anderson
Marilyn A. AndersonAffiliation: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia
Bio-protocol author page: a3954
 and Marie-Isabel Aguilar
Marie-Isabel AguilarAffiliation: Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia
Bio-protocol author page: a3955
date: 1/5/2017, 97 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2087.

Brief version appeared in Biochim Biophys Acta, Jun 2016
Examining the interaction of peptides with lipid bilayers to determine binding kinetics is often performed using surface plasmon resonance (SPR). Here we describe the technique of dual polarisation interferometry (DPI) that provides not only information on the kinetics of the peptide binding to the bilayer, but also how the peptide affects the lipid order of the bilayer.

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.

In vitro Assays for the Detection of Calreticulin Exposure, ATP and HMGB1 Release upon Cell Death

Featured protocol,  Authors: Yuting Ma
Yuting MaAffiliation 1: , Suzhou Institute of Systems Medicine, Suzhou, China
Affiliation 2: , Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
For correspondence: yuting_ma1984@163.com
Bio-protocol author page: a3918
 and Heng Yang
Heng YangAffiliation 1: , Suzhou Institute of Systems Medicine, Suzhou, China
Affiliation 2: , Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
Bio-protocol author page: a3919
date: 12/20/2016, 218 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2076.

Brief version appeared in Cancer Res, Sep 2015
Accumulating evidence is revealing the essential role of immune system in cancer treatment. Certain chemotherapeutic drugs can potently induce the release of ‘cell death associated molecular patterns’ (CDAMPs), which accompanies cancer cell demise. CDAMPs can engage corresponding receptors on immune cells and stimulate immune responses to achieve long-term tumor control (Ma et al., 2013; Ma et al., 2014; Yang et al., 2015). Among reported CDAMPs, calreticulin (CALR), ATP and HMGB1 are well known for their immune-stimulatory effect. Here we describe the assays that we applied to measure cell death and these CDAMPs. Briefly, cell death can be analyzed by co-staining of 4’,6-diamidino-2-phenylindole (DAPI) with 3,3’-Dihexyloxacarbocyanine Iodide [DiOC6(3)] or Annexin V. CALR exposure on the cell membrane can be detected by flow cytometry. ATP and HMGB1 release can be quantified by luminescence assay and ELISA assay respectively.

Activity-based Pull-down of Proteolytic Standard and Immunoproteasome Subunits

Featured protocol,  Authors: Tobias Baumann*
Tobias BaumannAffiliation: Comprehensive Pneumology Center (CPC), University Hospital, Ludwig-Maximilians University and Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Munich, Germany
Bio-protocol author page: a3909
Oliver Vosyka*
Oliver VosykaAffiliation: Comprehensive Pneumology Center (CPC), University Hospital, Ludwig-Maximilians University and Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Munich, Germany
Bio-protocol author page: a3910
Bogdan I. Florea
Bogdan I. FloreaAffiliation: Department of Bio-organic Synthesis, Leiden University, Leiden, The Netherlands
Bio-protocol author page: a3911
Hermen S. Overkleeft
Hermen S. OverkleeftAffiliation: Department of Bio-organic Synthesis, Leiden University, Leiden, The Netherlands
Bio-protocol author page: a3912
Silke Meiners
Silke MeinersAffiliation: Comprehensive Pneumology Center (CPC), University Hospital, Ludwig-Maximilians University and Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Munich, Germany
Bio-protocol author page: a3913
 and Ilona E. Kammerl
Ilona E. KammerlAffiliation: Comprehensive Pneumology Center (CPC), University Hospital, Ludwig-Maximilians University and Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Munich, Germany
For correspondence: ilona.kammerl@helmholtz-muenchen.de
Bio-protocol author page: a3914
 (*contributed equally to this work) date: 12/20/2016, 212 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2073.

Brief version appeared in Cell Death Differ, Jun 2016
Activity-based probes (ABP) are small organic molecules that irreversibly bind to the active center of a specific enzyme family and may be coupled to a fluorophore or an affinity tag (Li et al., 2013). Here, we describe a method to pull-down active catalytic standard and immunoproteasome subunits in cell lysates using the biotinylated, proteasome-specific ABP Biotin-Epoxomicin (Bio-EP). Covalent labeling of the active catalytic subunits with Bio-EP is followed by a pull-down using streptavidin-coated beads. After elution from the beads, enriched subunits may be detected via Western blot, tandem mass spectrometry (Li et al., 2013), or alternative techniques.

Quantitative Determination of Ascorbate from the Green Alga Chlamydomonas reinhardtii by HPLC

Featured protocol,  Authors: László Kovács
László KovácsAffiliation: Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary
Bio-protocol author page: a3895
André Vidal-Meireles
André Vidal-MeirelesAffiliation: Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary
Bio-protocol author page: a3896
Valéria Nagy
Valéria NagyAffiliation: Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary
Bio-protocol author page: a3897
 and Szilvia Z. Tóth
Szilvia Z. TóthAffiliation: Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary
For correspondence: toth.szilviazita@brc.mta.hu
Bio-protocol author page: a3898
date: 12/20/2016, 161 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2067.

Brief version appeared in Plant Cell Environ, Jul 2016
Ascorbate (Asc, also called vitamin C) is of vital importance to the cellular functions of both animals and plants. During evolution, Asc has become one of the most abundant metabolites in seed plants; however, Asc contents in cyanobacteria, green algae and bryophytes are very low. Here we describe a sensitive and reliable HPLC method for the quantitative determination of cellular Asc content in the green alga Chlamydomonas reinhardtii.

Expression, Purification and Crystallization of Recombinant Arabidopsis Monogalactosyldiacylglycerol Synthase (MGD1)

Featured protocol,  Authors: Joana Rocha
Joana RochaAffiliation: CERMAV-CNRS, University of Grenoble, Grenoble, France
Bio-protocol author page: a3887
Valerie Chazalet
Valerie ChazaletAffiliation: CERMAV-CNRS, University of Grenoble, Grenoble, France
Bio-protocol author page: a3888
 and Christelle Breton
Christelle BretonAffiliation: CERMAV-CNRS, University of Grenoble, Grenoble, France
For correspondence: breton@cermav.cnrs.fr
Bio-protocol author page: a3889
date: 12/20/2016, 148 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2064.

Brief version appeared in Plant J, Mar 2016
In plant cells, galactolipids are predominant, representing up to 50% of the lipid content in photosynthetic tissues. Galactolipid synthesis is initiated by MGDG synthases (MGDs), which use UDP-galactose as a donor sugar and diacylglycerol (DAG) as acceptor, to form monogalactosyldiacylglycerol (MGDG). This protocol is used to produce a recombinant form of Arabidopsis thaliana (A. thaliana) monogalactosyldiacylglycerol synthase 1 (MGD1) protein, in Escherichia coli (E. coli), using a two-step chromatographic purification procedure. The protein is easily expressed and purified to milligram quantities, suitable for biochemical and structural studies. The crystallization of MGD1 is also described.

Detection of Reactive Oxygen Species in Oryza sativa L. (Rice)

Featured protocol,  Authors: Navdeep Kaur
Navdeep KaurAffiliation: Department of Biotechnology, Guru Nanak Dev University, Amritsar, Punjab, India
Bio-protocol author page: a3877
Isha Sharma
Isha SharmaAffiliation: Department of Biotechnology, Guru Nanak Dev University, Amritsar, Punjab, India
Bio-protocol author page: a3878
Kamal Kirat
Kamal KiratAffiliation: Department of Biotechnology, Guru Nanak Dev University, Amritsar, Punjab, India
Bio-protocol author page: a3879
 and Pratap Kumar Pati
Pratap Kumar PatiAffiliation: Department of Biotechnology, Guru Nanak Dev University, Amritsar, Punjab, India
For correspondence: pkpati@yahoo.com
Bio-protocol author page: a3876
date: 12/20/2016, 199 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2061.

Brief version appeared in BMC Plant Biol, Jun 2016
Superoxide ions (O2-) and hydrogen peroxide (H2O2) are the reactive oxygen species (ROS) that play a significant role in regulation of many plant processes. The level of O2- ions is determined qualitatively using nitrobluetetrazolium (NBT) assay while the H2O2 is qualitatively estimated using 3,3-diaminobenzidine (DAB) and 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) assay. Further the aqueous content of H2O2 is estimated quantitatively using ferrous oxidation-xylenol orange (FOX) assay.

Protocol for Increasing Carotenoid Levels in the Roots of Citrus Plants

Featured protocol,  Authors: Matías Manzi
Matías ManziAffiliation 1: Ecofisiologia i Biotecnologia, Dept. Ciencies Agraries i del Medi Natural, Universitat Jaume I, Castello de la Plana, Spain
Affiliation 2: Fertilidad de Suelos, Dept. Suelos y Agua, Facultad de Agronomía, Universidad de la República, Salto, Uruguay
Bio-protocol author page: a3922
Marta Pitarch-Bielsa
Marta Pitarch-BielsaAffiliation: Ecofisiologia i Biotecnologia, Dept. Ciencies Agraries i del Medi Natural, Universitat Jaume I, Castello de la Plana, Spain
Bio-protocol author page: a3921
Vicent Arbona
Vicent ArbonaAffiliation: Ecofisiologia i Biotecnologia, Dept. Ciencies Agraries i del Medi Natural, Universitat Jaume I, Castello de la Plana, Spain
Bio-protocol author page: a3923
 and Aurelio Gómez-Cadenas
Aurelio Gómez-CadenasAffiliation: Ecofisiologia i Biotecnologia, Dept. Ciencies Agraries i del Medi Natural, Universitat Jaume I, Castello de la Plana, Spain
For correspondence: aurelio.gomez@uji.es
Bio-protocol author page: a3924
date: 12/20/2016, 153 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2077.

Brief version appeared in Plant Sci, Nov 2016
Carotenoids in plants play several key functions such as acting as light-harvesters, antioxidants (Lado et al., 2016) or being precursors of strigolactones, abscisic acid, volatiles and other signaling compounds (Arbona et al., 2013). Although those functions are well-known in light-exposed tissues, information in belowground organs is limited because of reduced abundance of these pigments. In order to better understand the role of carotenoids in roots, we developed a methodology to increase the abundance of these pigments in underground tissues. We took advantage of the fact that citrus roots exposed to light develop pigmentation in order to increase the carotenoid content. Therefore, here we describe a simple method to increase carotenoids in citrus roots.

Bacterial Intracellular Sodium Ion Measurement using CoroNa Green

Authors: Yusuke V. Morimoto
Yusuke V. MorimotoAffiliation 1: Quantitative Biology Center, RIKEN, Suita, Osaka, Japan
Affiliation 2: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan
Bio-protocol author page: a3969
Keiichi Namba
Keiichi NambaAffiliation 1: Quantitative Biology Center, RIKEN, Suita, Osaka, Japan
Affiliation 2: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan
Bio-protocol author page: a3970
 and Tohru Minamino
Tohru MinaminoAffiliation: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan
For correspondence: tohru@fbs.osaka-u.ac.jp
Bio-protocol author page: a3971
date: 1/5/2017, 109 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2092.

[Abstract] The bacterial flagellar type III export apparatus consists of a cytoplasmic ATPase complex and a transmembrane export gate complex, which are powered by ATP and proton motive force (PMF) across the cytoplasmic membrane, respectively, and transports flagellar component proteins from the cytoplasm to the distal end of the growing flagellar structure ...

Measuring Oxidative Stress in Caenorhabditis elegans: Paraquat and Juglone Sensitivity Assays

Authors: Megan M. Senchuk
Megan M. SenchukAffiliation: Laboratory of Aging and Neurodegenerative Disease (LAND), Center for Neurodegenerative Science, Van Andel Research Institute, Grand Rapids, USA
Bio-protocol author page: a3949
Dylan J. Dues
Dylan J. Dues Affiliation: Laboratory of Aging and Neurodegenerative Disease (LAND), Center for Neurodegenerative Science, Van Andel Research Institute, Grand Rapids, USA
Bio-protocol author page: a3950
 and Jeremy M. Van Raamsdonk
Jeremy M. Van RaamsdonkAffiliation: Laboratory of Aging and Neurodegenerative Disease (LAND), Center for Neurodegenerative Science, Van Andel Research Institute, Grand Rapids, USA
For correspondence: jeremy.vanraamsdonk@vai.org
Bio-protocol author page: a3951
date: 1/5/2017, 106 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2086.

[Abstract] Oxidative stress has been proposed to be one of the main causes of aging and has been implicated in the pathogenesis of many diseases. Sensitivity to oxidative stress can be measured by quantifying survival following exposure to a reactive oxygen species (ROS)-generating compound such as paraquat or juglone. Sensitivity to oxidative stress is a balance ...

Cation (Ca2+ and Mn2+) Partitioning Assays with Intact Arabidopsis Chloroplasts

Authors: Anna Harms
Anna HarmsAffiliation: Biozentrum der LMU München, Department Biologie I, Munich, Germany
Bio-protocol author page: a3963
Iris Steinberger
Iris SteinbergerAffiliation: Biozentrum der LMU München, Department Biologie I, Munich, Germany
Bio-protocol author page: a2369
 and Anja Schneider
Anja SchneiderAffiliation: Biozentrum der LMU München, Department Biologie I, Munich, Germany
For correspondence: anja.schneider@lrz.uni-muenchen.de
Bio-protocol author page: a2371
date: 1/5/2017, 90 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2094.

[Abstract] Determination of the relative distribution of Ca2+ and Mn2+ is an important tool for analyzing mutants showing altered levels of calcium and/or manganese transporters in the chloroplast envelope or thylakoid membrane. The method described in this protocol allows quantitative analyses of the relative distribution of calcium and manganese ions between ...

In vitro Ubiquitin Dimer Formation Assay

Authors: Sheng Wang
Sheng WangAffiliation: Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada
Bio-protocol author page: a1483
Ling Cao
Ling CaoAffiliation 1: Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada
Affiliation 2: National Key laboratory of Crop Genetic Improvement, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China
Bio-protocol author page: a1482
 and Hong Wang
Hong WangAffiliation: Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada
For correspondence: hong.wang@usask.ca
Bio-protocol author page: a1485
date: 1/5/2017, 108 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2082.

[Abstract] The process of protein ubiquitination typically consists of three sequential steps to add an ubiquitin (Ub) or Ub chain to a substrate protein, requiring three different enzymes, ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin protein ligase (E3). Most E2s possess the classical E2 activity in forming E2-Ub complex ...

Examination of the Interaction between a Membrane Active Peptide and Artificial Bilayers by Dual Polarisation Interferometry

Authors: Jennifer A.E. Payne
Jennifer A.E. PayneAffiliation 1: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia
Affiliation 2: Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia
For correspondence: jennifer.payne@monash.edu
Bio-protocol author page: a3952
Tzong-Hsien Lee
Tzong-Hsien LeeAffiliation: Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia
Bio-protocol author page: a3953
Marilyn A. Anderson
Marilyn A. AndersonAffiliation: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia
Bio-protocol author page: a3954
 and Marie-Isabel Aguilar
Marie-Isabel AguilarAffiliation: Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia
Bio-protocol author page: a3955
date: 1/5/2017, 97 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2087.

[Abstract] Examining the interaction of peptides with lipid bilayers to determine binding kinetics is often performed using surface plasmon resonance (SPR). Here we describe the technique of dual polarisation interferometry (DPI) that provides not only information on the kinetics of the peptide binding to the bilayer, but also how the peptide affects the lipid ...

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 clipping of their tail domains. The proteolytic clipping ...

In vitro Assays for the Detection of Calreticulin Exposure, ATP and HMGB1 Release upon Cell Death

Authors: Yuting Ma
Yuting MaAffiliation 1: , Suzhou Institute of Systems Medicine, Suzhou, China
Affiliation 2: , Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
For correspondence: yuting_ma1984@163.com
Bio-protocol author page: a3918
 and Heng Yang
Heng YangAffiliation 1: , Suzhou Institute of Systems Medicine, Suzhou, China
Affiliation 2: , Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
Bio-protocol author page: a3919
date: 12/20/2016, 218 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2076.

[Abstract] Accumulating evidence is revealing the essential role of immune system in cancer treatment. Certain chemotherapeutic drugs can potently induce the release of ‘cell death associated molecular patterns’ (CDAMPs), which accompanies cancer cell demise. CDAMPs can engage corresponding receptors on immune cells and stimulate immune responses to achieve long-term ...

Activity-based Pull-down of Proteolytic Standard and Immunoproteasome Subunits

Authors: Tobias Baumann*
Tobias BaumannAffiliation: Comprehensive Pneumology Center (CPC), University Hospital, Ludwig-Maximilians University and Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Munich, Germany
Bio-protocol author page: a3909
Oliver Vosyka*
Oliver VosykaAffiliation: Comprehensive Pneumology Center (CPC), University Hospital, Ludwig-Maximilians University and Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Munich, Germany
Bio-protocol author page: a3910
Bogdan I. Florea
Bogdan I. FloreaAffiliation: Department of Bio-organic Synthesis, Leiden University, Leiden, The Netherlands
Bio-protocol author page: a3911
Hermen S. Overkleeft
Hermen S. OverkleeftAffiliation: Department of Bio-organic Synthesis, Leiden University, Leiden, The Netherlands
Bio-protocol author page: a3912
Silke Meiners
Silke MeinersAffiliation: Comprehensive Pneumology Center (CPC), University Hospital, Ludwig-Maximilians University and Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Munich, Germany
Bio-protocol author page: a3913
 and Ilona E. Kammerl
Ilona E. KammerlAffiliation: Comprehensive Pneumology Center (CPC), University Hospital, Ludwig-Maximilians University and Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Munich, Germany
For correspondence: ilona.kammerl@helmholtz-muenchen.de
Bio-protocol author page: a3914
 (*contributed equally to this work) date: 12/20/2016, 212 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2073.

[Abstract] Activity-based probes (ABP) are small organic molecules that irreversibly bind to the active center of a specific enzyme family and may be coupled to a fluorophore or an affinity tag (Li et al., 2013). Here, we describe a method to pull-down active catalytic standard and immunoproteasome subunits in cell lysates using the biotinylated, proteasome-specific ...

Quantitative Determination of Ascorbate from the Green Alga Chlamydomonas reinhardtii by HPLC

Authors: László Kovács
László KovácsAffiliation: Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary
Bio-protocol author page: a3895
André Vidal-Meireles
André Vidal-MeirelesAffiliation: Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary
Bio-protocol author page: a3896
Valéria Nagy
Valéria NagyAffiliation: Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary
Bio-protocol author page: a3897
 and Szilvia Z. Tóth
Szilvia Z. TóthAffiliation: Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary
For correspondence: toth.szilviazita@brc.mta.hu
Bio-protocol author page: a3898
date: 12/20/2016, 161 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2067.

[Abstract] Ascorbate (Asc, also called vitamin C) is of vital importance to the cellular functions of both animals and plants. During evolution, Asc has become one of the most abundant metabolites in seed plants; however, Asc contents in cyanobacteria, green algae and bryophytes are very low. Here we describe a sensitive and reliable HPLC method for the quantitative ...

Expression, Purification and Crystallization of Recombinant Arabidopsis Monogalactosyldiacylglycerol Synthase (MGD1)

Authors: Joana Rocha
Joana RochaAffiliation: CERMAV-CNRS, University of Grenoble, Grenoble, France
Bio-protocol author page: a3887
Valerie Chazalet
Valerie ChazaletAffiliation: CERMAV-CNRS, University of Grenoble, Grenoble, France
Bio-protocol author page: a3888
 and Christelle Breton
Christelle BretonAffiliation: CERMAV-CNRS, University of Grenoble, Grenoble, France
For correspondence: breton@cermav.cnrs.fr
Bio-protocol author page: a3889
date: 12/20/2016, 148 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2064.

[Abstract] In plant cells, galactolipids are predominant, representing up to 50% of the lipid content in photosynthetic tissues. Galactolipid synthesis is initiated by MGDG synthases (MGDs), which use UDP-galactose as a donor sugar and diacylglycerol (DAG) as acceptor, to form monogalactosyldiacylglycerol (MGDG). This protocol is used to produce a recombinant ...
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[Bio101] Bradford Protein Assay

Author: Fanglian He date: 3/20/2011, 74280 views, 4 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, 59572 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 ...

[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, 38791 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....

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, 38163 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 ...

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, 30523 views, 15 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, 29587 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] 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, 29437 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 ...

[Bio101] Coomassie Blue Staining

Author: Fanglian He date: 6/5/2011, 28146 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, 28132 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, 22728 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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