Protocols in Current Issue
    DNA-free Genome Editing of Chlamydomonas reinhardtii Using CRISPR and Subsequent Mutant Analysis
    Authors:  Jihyeon Yu, Kwangryul Baek, EonSeon Jin and Sangsu Bae, date: 06/05/2017, view: 6725, Q&A: 0
    [Abstract] We successfully introduced targeted knock-out of gene of interest in Chlamydomonas reinhardtii by using DNA-free CRISPR. In this protocol, the detailed procedures of an entire workflow cover from the initial target selection of CRISPR to the mutant analysis using next generation sequencing (NGS) technology. Furthermore, we introduce a ...
    Indirect Immunofluorescence Assay in Chlamydomonas reinhardtii
    Authors:  Takashi Yamano and Hideya Fukuzawa, date: 07/05/2016, view: 6729, Q&A: 0
    [Abstract] Determining the protein localization is essential to elucidate its in vivo function. Fluorescence-tagged proteins are widely used for it, but it is sometimes difficult to express tagged proteins in Chlamydomonas. Alternatively, indirect immunofluorescence assay is also one of the widely used methods and many reports determining ...
    Purification of Rubisco from Chlamydomonas reinhardtii
    [Abstract] Chlamydomonas reinhardtii is a model organism for chloroplast studies. Besides other convenient features, the feasibility of chloroplast genome transformation distinguishes this unicellular alga as ideal for the manipulation of chloroplastic gene expression aiming biotechnological goals, such as improved biofuel and biomass production. ...
    Assay of the Carboxylase Activity of Rubisco from Chlamydomonas reinhardtii
    [Abstract] The performance of the carbon-fixing enzyme, ribulose 1, 5-bisphosphate carboxylase/oxygenase (EC, Rubisco), controls biomass accumulation in green plants, algae and most autotrophic bacteria. In particular, the carboxylase activity of Rubisco incorporates carbon from CO2 to ribulose 1, 5-bisphosphate (RuBP) producing two ...
    Pyruvate:ferredoxin Oxidoreductase (PFR1) Activity Assays Using Methyl Viologen as Artificial Electron Acceptor
    Author:  Jens Noth, date: 09/05/2013, view: 8276, Q&A: 0
    [Abstract] Here we describe the activity measurements of heterologous expressed pyruvate:ferredoxin oxidoreductase (Noth et al., 2013) (Noth et al.,2013) from Chlamydomonas reinhardtii. This enzyme catalyzes the reversible reaction (I) from pyruvate to acetyl CoA and CO2 generating low potential electrons which are in ...
    Heterologous Production and Anaerobic Purification of His- and StrepII-tagged Recombinant Proteins
    Author:  Jens Noth, date: 09/05/2013, view: 10378, Q&A: 0
    [Abstract] This protocol describes the heterologous expression and purification of proteins related to anoxic hydrogen production of Chlamydomonas reinhardtii (Noth et al., 2013). For this, the bacterial expression hosts Escherichia coli BL21 (DE3) ΔiscR (Akhtar MK et al., 2008) and Clostridium acetobutylicum ...
    Western Blot Analysis of Chloroplast HSP70B in Chlorella Species
    Authors:  Stephka Chankova, Zhana Mitrovska and Nadezhda Yurina, date: 08/05/2013, view: 6446, Q&A: 0
    [Abstract] Western blotting allows for the specific detection of proteins by an antibody of interest. This protocol utilizes isolation of total proteins protocol for Chlorella vulgaris prior to gel electrophoresis. After electrophoresis, the selected antibodies are used to detect and quantify levels of chloroplast HSP70B.
    Generation of Polyclonal Specific Antibodies
    Authors:  Maureen Wirschell and Mary E. Porter, date: 06/05/2013, view: 7278, Q&A: 0
    [Abstract] Generation of antibodies specific for a protein of interest is a common method in many disciplines. This protocol details the steps in production of a polyclonal antibody in rabbits using a bacterially expressed fusion protein as an antigen. The protocol is generated based on data presented in Wirschell et al.(2013).
    35S pulse Labelling of Chlamydomonas Chloroplast Proteins
    Authors:  Alexandra-Viola Bohne, Christian Schwarz and Joerg Nickelsen, date: 06/05/2013, view: 7021, Q&A: 0
    [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 ...
    2D Diagonal Redox SDS-PAGE of Proteins
    Authors:  Christian Schwarz and Joerg Nickelsen, date: 06/05/2013, view: 10429, Q&A: 0
    [Abstract] 2D diagonal redox SDS-PAGE of proteins is used to detect intramolecular or intermolecular disulfide bridges using Chlamydomonas in this example (Stroeher and Dietz, 2008; Schwarz et al., 2012). Both dimensions consist of a conventional SDS-PAGE, except that the sample buffer for the first dimension lacks a reducing agent. ...

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