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    Protocols in Current Issue
    Isolation and Detection of the Chlorophyll Catabolite Hydroxylating Activity from Capsicum annuum Chromoplasts
    Authors:  Mareike Hauenstein and Stefan Hörtensteiner, date: 09/20/2017, view: 724, Q&A: 0
    [Abstract] Hydroxylation of chlorophyll catabolites at the so-called C32 position (Hauenstein et al., 2016) is commonly found in all plant species analyzed to date. Here we describe an in vitro hydroxylation assay using Capsicum annuum chromoplast membranes as a source of the hydroxylating activity, which converts the ...
    Ubiquitin Proteasome Activity Measurement in Total Plant Extracts
    Authors:  Suayib Üstün and Frederik Börnke, date: 09/05/2017, view: 724, Q&A: 0
    [Abstract] The fine-tuned balance of protein level, conformation and location within the cell is vital for the dynamic changes required for a cell to respond to a given stimulus. This requires the regulated turnover of damaged or short-lived proteins through the ubiquitin proteasome system (UPS). Thus, the protease activity of the proteasome is adjusted to ...
    Separation of Plant 6-Phosphogluconate Dehydrogenase (6PGDH) Isoforms by Non-denaturing Gel Electrophoresis
    [Abstract] 6-Phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) catalyzes the third and irreversible reaction of the pentose phosphate pathway (PPP). It carries out the oxidative decarboxylation of the 6-phosphogluconate to yield ribulose-5-phosphate, carbon dioxide and NADPH. In higher plants, 6PGDH has several subcellular localizations including cytosol, ...
    Extraction and Activity of O-acetylserine(thiol)lyase (OASTL) from Microalga Chlorella sorokiniana
    Authors:  Giovanna Salbitani and Simona Carfagna, date: 06/20/2017, view: 959, Q&A: 0
    [Abstract] O-acetylserine(thiol)lyase (OASTL) is an enzyme catalysing the reaction of inorganic sulphide with O-acetylserine to form the S-containing amino acid L-cysteine. Here we describe an improved protocol to evaluate the activity of this enzyme from the microalga Chlorella sorokiniana. It is a colorimetric assay based on the reaction between ...
    Analyses of Root-secreted Acid Phosphatase Activity in Arabidopsis
    Authors:  Liangsheng Wang and Dong Liu, date: 04/05/2017, view: 1433, Q&A: 0
    [Abstract] Induction and secretion of acid phosphatase (APase) is a universal adaptive response of higher plants to low-phosphate stress (Tran et al., 2010). The intracellular APases are likely involved in the remobilization and recycling of phosphate (Pi) from intracellular Pi reserves, whereas the extracellular or secreted APases are believed to ...
    Ribosomal RNA N-glycosylase Activity Assay of Ribosome-inactivating Proteins
    Authors:  Rosario Iglesias, Lucía Citores and José M. Ferreras, date: 03/20/2017, view: 1976, Q&A: 0
    [Abstract] Ribosome-inactivating proteins (RIPs) are enzymes that irreversibly inactivate ribosomes as a consequence of their N-glycosylase (EC 3.2.2.22) activity. The enzyme cleaves the N-glycosidic bond between the adenine No. 4324 from the 28S rRNA and its ribose in rat ribosomes (or the equivalent adenine in sensitive ribosomes from other organisms). ...
    In Gel Kinase Assay
    Authors:  Gaston A. Pizzio and Pedro L. Rodriguez, date: 03/05/2017, view: 2122, Q&A: 0
    [Abstract] Proper spatiotemporal regulation of protein phosphorylation in cells and tissues is required for normal development and homeostasis. We present the protocol ‘In Gel Kinase Assay’, which is useful for protein kinase activity measurements from crude protein extracts. We have successfully used ‘In Gel Kinase Assay’ protocol to show that the ...
    Direct Visualization and Quantification of the Actin Nucleation and Elongation Events in vitro by TIRF Microscopy
    Authors:  Yuxiang Jiang and Shanjin Huang, date: 03/05/2017, view: 1478, Q&A: 0
    [Abstract] Total internal reflection fluorescence (TIRF) microscopy is a powerful tool for visualizing the dynamics of actin filaments at single-filament resolution in vitro. Thanks to the development of various fluorescent probes, we can easily monitor all kinds of events associated with actin dynamics, including nucleation, elongation, bundling, ...
    Two-electrode Voltage-clamp Recordings in Xenopus laevis Oocytes:Reconstitution of Abscisic Acid Activation of SLAC1 Anion Channel via PYL9 ABA Receptor
    Authors:  Cun Wang, Jingbo Zhang and Julian I. Schroeder, date: 01/20/2017, view: 1925, Q&A: 0
    [Abstract] Two-Electrode Voltage-Clamp (TEVC) recording in Xenopus laevis oocytes provides a powerful method to investigate the functions and regulation of ion channel proteins. This approach provides a well-known tool to characterize ion channels or transporters expressed in Xenopus laevis oocytes. The plasma membrane of the oocyte is ...
    In vitro Ubiquitin Dimer Formation Assay
    Authors:  Sheng Wang, Ling Cao and Hong Wang, date: 01/05/2017, view: 1722, Q&A: 0
    [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 ...