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Rubisco Extraction and Purification from Diatoms

Authors: Jodi N. Young
Jodi N. YoungAffiliation: Department of Oceanography, University of Washington, Seattle, USA
For correspondence: youngjn@uw.edu
Bio-protocol author page: a4026
Ana M. C. Heureux
Ana M. C. HeureuxAffiliation: Department of Earth Sciences, University of Oxford, Oxford, UK
Bio-protocol author page: a4027
Rosalind E. M. Rickaby
Rosalind E. M. RickabyAffiliation: Department of Earth Sciences, University of Oxford, Oxford, UK
Bio-protocol author page: a4028
François M. M. Morel
François M. M. MorelAffiliation: Department of Geosciences, Princeton University, Princeton, USA
Bio-protocol author page: a4029
Spencer M. Whitney
Spencer M. WhitneyAffiliation: Plant Science Division, Research School of Biology, the Australian National University, Canberra, Australia
Bio-protocol author page: a4030
 and Robert E. Sharwood
Robert E. SharwoodAffiliation: Plant Science Division, Research School of Biology, the Australian National University, Canberra, Australia
Bio-protocol author page: a4031
date: 3/20/2017, 668 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2191.

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

Chlorophyll Fluorescence Measurements in Arabidopsis Plants Using a Pulse-amplitude-modulated (PAM) Fluorometer

Authors: Reiko Motohashi
Reiko MotohashiAffiliation: Agriculture Department, Shizuoka University, Shizuoka, Japan
For correspondence: armotoh@ipc.shizuoka.ac.jp
Bio-protocol author page: a2179
 and Fumiyoshi Myouga
Fumiyoshi MyougaAffiliation: RIKEN Center for Sustainable Resource Science, Yokohama, Japan
Bio-protocol author page: a2181
date: 5/5/2015, 4627 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1464.

[Abstract] In this protocol, to analyze PSII activity in photosynthesis, we measure the Fv/Fm (Fv=Fm ± Fo) value (Fo and Fm are the minimum and maximum values of chlorophyll fluorescence of dark-adapted leaves, respectively). Fv/Fm is a reliable marker of photo- inhibition (Krause et al., 1988). Chlorophyll fluorescence in leaves was measured at room temperature ...

Measurement of Chlorophyll a and Carotenoids Concentration in Cyanobacteria

Authors: Tomáš Zavřel
Tomáš ZavřelAffiliation: Department of Adaptation Biotechnologies, Global Change Research Centre, Academy of Science of the Czech Republic, Drásov, Czech Republic
For correspondence: zavrel.t@czechglobe.cz
Bio-protocol author page: a2192
Maria A. Sinetova
Maria A. SinetovaAffiliation: Laboratory of Intracellular Regulation, Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia
Bio-protocol author page: a243
 and Jan Červený
Jan ČervenýAffiliation: Department of Adaptation Biotechnologies, Global Change Research Centre, Academy of Science of the Czech Republic, Drásov, Czech Republic
Bio-protocol author page: a2193
date: 5/5/2015, 6599 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1467.

[Abstract] This is a protocol for precise measurement of chlorophyll a and total carotenoid concentrations in cyanobacteria cells. Cellular chlorophyll concentration is one of the central physiological parameters, routinely followed in many research areas ranging from stress physiology to biotechnology. Carotenoids concentration is often related to cellular stress ...

Detection and Quantification of Heme and Chlorophyll Precursors Using a High Performance Liquid Chromatography (HPLC) System Equipped with Two Fluorescence Detectors

Authors: Jan Pilný
Jan PilnýAffiliation: Institute of Microbiology, Academy of Sciences, Třeboň, Czech Republic
Bio-protocol author page: a1970
Jana Kopečná
Jana KopečnáAffiliation: Institute of Microbiology, Academy of Sciences, Třeboň, Czech Republic
Bio-protocol author page: a86
Judith Noda
Judith NodaAffiliation: Institute of Microbiology, Academy of Sciences, Třeboň, Czech Republic
Bio-protocol author page: a1971
 and Roman Sobotka
Roman SobotkaAffiliation: Institute of Microbiology, Academy of Sciences, Třeboň, Czech Republic
For correspondence: sobotka@alga.cz
Bio-protocol author page: a84
date: 2/5/2015, 3252 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1390.

[Abstract] Intermediates of tetrapyrrole biosynthetic pathway are low-abundant compounds, and their quantification is usually difficult, time consuming and requires large amounts of input material. Here, we describe a method allowing fast and accurate quantification of almost all intermediates of the heme and chlorophyll biosynthesis, including mono-vinyl and ...

Analysis of Malondialdehyde, Chlorophyll Proline, Soluble Sugar, and Glutathione Content in Arabidopsis seedling

Authors: Zhijin Zhang
Zhijin ZhangAffiliation: Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou, China
Bio-protocol author page: a684
 and Rongfeng Huang
Rongfeng HuangAffiliation: Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou, China
For correspondence: rfhuang@caas.cn
Bio-protocol author page: a362
date: 7/20/2013, 11118 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.817.

[Abstract] The protocol has four sub-protocols, which are about the measurement of malondialdehyde, chlorophyll proline, soluble sugar, and glutathione content, respectively, in Arabidopsis seedling by using spectrophotometer. These methods are simple, effective and reproducible, which will help the researchers who are not familiar with these approaches, quickly ...

35S pulse Labelling of Chlamydomonas Chloroplast Proteins

Authors: Alexandra-Viola Bohne
Alexandra-Viola BohneAffiliation: Molecular Plant Science, Ludwig-Maximilians-Universität, Munich, Germany
Bio-protocol author page: a615
Christian Schwarz
Christian SchwarzAffiliation: Molecular Plant Science, Ludwig-Maximilians-Universität, Munich, Germany
Bio-protocol author page: a609
 and Joerg Nickelsen
Joerg NickelsenAffiliation: Molecular Plant Science, Ludwig-Maximilians-Universität, Munich, Germany
For correspondence: joerg.nickelsen@lrz.uni-muenchen.de
Bio-protocol author page: a610
date: 6/5/2013, 3952 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.783.

[Abstract] 35S pulse labelling of proteins is used to attach a radioactive label to newly synthesized proteins, as sulfur is an element that is mainly present in proteins (Fleischmann and Rochaix 1999). Depending on your organism’s uptake mechanisms you need cysteine, methionine or sulfuric acid as a source of radioactive sulfur. This example uses Chlamydomonas ...

Radiolabeling of Chlorophyll by [14C]Glutamic Acid in vivo and Relative Quantification of Labeled Chlorophyll by Using Thin Layer Chromatography (TLC)

Authors: Roman Sobotka
Roman SobotkaAffiliation: Department of Phototrophic Microorganisms, Institute of Microbiology ASCR, Trebon, Czech Republic
For correspondence: sobotka@alga.cz
Bio-protocol author page: a84
Luděk Kořený
Luděk KořenýAffiliation: Department of Molecular Parasitology, Institute of Parasitology, Biology Centre ASCR, Ceske Budejovice, Czech Republic
Bio-protocol author page: a85
Jana Kopečná
Jana KopečnáAffiliation: Department of Phototrophic Microorganisms, Institute of Microbiology ASCR, Trebon, Czech Republic
Bio-protocol author page: a86
 and Miroslav Oborník
Miroslav OborníkAffiliation: Department of Molecular Parasitology, Institute of Parasitology, Biology Centre ASCR, Ceske Budejovice, Czech Republic
Bio-protocol author page: a87
date: 8/20/2012, 3989 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.249.

[Abstract] This is an accurate method to assess the rate of chlorophyll biosynthesis in vivo in cyanobacteria. Given that labeled glutamate is used as the very early precursor of chlorophyll together with a short pulse of labeling (30 min), this method provides information about the metabolic flow through the whole chlorophyll biosynthetic pathway on a short ...
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Analysis of Malondialdehyde, Chlorophyll Proline, Soluble Sugar, and Glutathione Content in Arabidopsis seedling

Authors: Zhijin Zhang
Zhijin ZhangAffiliation: Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou, China
Bio-protocol author page: a684
 and Rongfeng Huang
Rongfeng HuangAffiliation: Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou, China
For correspondence: rfhuang@caas.cn
Bio-protocol author page: a362
date: 7/20/2013, 11118 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.817.

[Abstract] The protocol has four sub-protocols, which are about the measurement of malondialdehyde, chlorophyll proline, soluble sugar, and glutathione content, respectively, in Arabidopsis seedling by using spectrophotometer. These methods are simple, effective and reproducible, which will help the researchers ...

Measurement of Chlorophyll a and Carotenoids Concentration in Cyanobacteria

Authors: Tomáš Zavřel
Tomáš ZavřelAffiliation: Department of Adaptation Biotechnologies, Global Change Research Centre, Academy of Science of the Czech Republic, Drásov, Czech Republic
For correspondence: zavrel.t@czechglobe.cz
Bio-protocol author page: a2192
Maria A. Sinetova
Maria A. SinetovaAffiliation: Laboratory of Intracellular Regulation, Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia
Bio-protocol author page: a243
 and Jan Červený
Jan ČervenýAffiliation: Department of Adaptation Biotechnologies, Global Change Research Centre, Academy of Science of the Czech Republic, Drásov, Czech Republic
Bio-protocol author page: a2193
date: 5/5/2015, 6599 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1467.

[Abstract] This is a protocol for precise measurement of chlorophyll a and total carotenoid concentrations in cyanobacteria cells. Cellular chlorophyll concentration is one of the central physiological parameters, routinely followed in many research areas ranging from stress physiology to biotechnology. Carotenoids ...

Chlorophyll Fluorescence Measurements in Arabidopsis Plants Using a Pulse-amplitude-modulated (PAM) Fluorometer

Authors: Reiko Motohashi
Reiko MotohashiAffiliation: Agriculture Department, Shizuoka University, Shizuoka, Japan
For correspondence: armotoh@ipc.shizuoka.ac.jp
Bio-protocol author page: a2179
 and Fumiyoshi Myouga
Fumiyoshi MyougaAffiliation: RIKEN Center for Sustainable Resource Science, Yokohama, Japan
Bio-protocol author page: a2181
date: 5/5/2015, 4627 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1464.

[Abstract] In this protocol, to analyze PSII activity in photosynthesis, we measure the Fv/Fm (Fv=Fm ± Fo) value (Fo and Fm are the minimum and maximum values of chlorophyll fluorescence of dark-adapted leaves, respectively). Fv/Fm is a reliable marker of photo- inhibition (Krause et al., 1988). Chlorophyll fluorescence ...

Radiolabeling of Chlorophyll by [14C]Glutamic Acid in vivo and Relative Quantification of Labeled Chlorophyll by Using Thin Layer Chromatography (TLC)

Authors: Roman Sobotka
Roman SobotkaAffiliation: Department of Phototrophic Microorganisms, Institute of Microbiology ASCR, Trebon, Czech Republic
For correspondence: sobotka@alga.cz
Bio-protocol author page: a84
Luděk Kořený
Luděk KořenýAffiliation: Department of Molecular Parasitology, Institute of Parasitology, Biology Centre ASCR, Ceske Budejovice, Czech Republic
Bio-protocol author page: a85
Jana Kopečná
Jana KopečnáAffiliation: Department of Phototrophic Microorganisms, Institute of Microbiology ASCR, Trebon, Czech Republic
Bio-protocol author page: a86
 and Miroslav Oborník
Miroslav OborníkAffiliation: Department of Molecular Parasitology, Institute of Parasitology, Biology Centre ASCR, Ceske Budejovice, Czech Republic
Bio-protocol author page: a87
date: 8/20/2012, 3989 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.249.

[Abstract] This is an accurate method to assess the rate of chlorophyll biosynthesis in vivo in cyanobacteria. Given that labeled glutamate is used as the very early precursor of chlorophyll together with a short pulse of labeling (30 min), this method provides information about the metabolic flow through the ...

35S pulse Labelling of Chlamydomonas Chloroplast Proteins

Authors: Alexandra-Viola Bohne
Alexandra-Viola BohneAffiliation: Molecular Plant Science, Ludwig-Maximilians-Universität, Munich, Germany
Bio-protocol author page: a615
Christian Schwarz
Christian SchwarzAffiliation: Molecular Plant Science, Ludwig-Maximilians-Universität, Munich, Germany
Bio-protocol author page: a609
 and Joerg Nickelsen
Joerg NickelsenAffiliation: Molecular Plant Science, Ludwig-Maximilians-Universität, Munich, Germany
For correspondence: joerg.nickelsen@lrz.uni-muenchen.de
Bio-protocol author page: a610
date: 6/5/2013, 3952 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.783.

[Abstract] 35S pulse labelling of proteins is used to attach a radioactive label to newly synthesized proteins, as sulfur is an element that is mainly present in proteins (Fleischmann and Rochaix 1999). Depending on your organism’s uptake mechanisms you need cysteine, methionine or sulfuric acid as a source of ...

Detection and Quantification of Heme and Chlorophyll Precursors Using a High Performance Liquid Chromatography (HPLC) System Equipped with Two Fluorescence Detectors

Authors: Jan Pilný
Jan PilnýAffiliation: Institute of Microbiology, Academy of Sciences, Třeboň, Czech Republic
Bio-protocol author page: a1970
Jana Kopečná
Jana KopečnáAffiliation: Institute of Microbiology, Academy of Sciences, Třeboň, Czech Republic
Bio-protocol author page: a86
Judith Noda
Judith NodaAffiliation: Institute of Microbiology, Academy of Sciences, Třeboň, Czech Republic
Bio-protocol author page: a1971
 and Roman Sobotka
Roman SobotkaAffiliation: Institute of Microbiology, Academy of Sciences, Třeboň, Czech Republic
For correspondence: sobotka@alga.cz
Bio-protocol author page: a84
date: 2/5/2015, 3252 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1390.

[Abstract] Intermediates of tetrapyrrole biosynthetic pathway are low-abundant compounds, and their quantification is usually difficult, time consuming and requires large amounts of input material. Here, we describe a method allowing fast and accurate quantification of almost all intermediates of the heme and ...

Rubisco Extraction and Purification from Diatoms

Authors: Jodi N. Young
Jodi N. YoungAffiliation: Department of Oceanography, University of Washington, Seattle, USA
For correspondence: youngjn@uw.edu
Bio-protocol author page: a4026
Ana M. C. Heureux
Ana M. C. HeureuxAffiliation: Department of Earth Sciences, University of Oxford, Oxford, UK
Bio-protocol author page: a4027
Rosalind E. M. Rickaby
Rosalind E. M. RickabyAffiliation: Department of Earth Sciences, University of Oxford, Oxford, UK
Bio-protocol author page: a4028
François M. M. Morel
François M. M. MorelAffiliation: Department of Geosciences, Princeton University, Princeton, USA
Bio-protocol author page: a4029
Spencer M. Whitney
Spencer M. WhitneyAffiliation: Plant Science Division, Research School of Biology, the Australian National University, Canberra, Australia
Bio-protocol author page: a4030
 and Robert E. Sharwood
Robert E. SharwoodAffiliation: Plant Science Division, Research School of Biology, the Australian National University, Canberra, Australia
Bio-protocol author page: a4031
date: 3/20/2017, 668 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2191.

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