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In vitro Reconstitution Assays of Arabidopsis 20S Proteasome

Authors: Yanjun Li, Di Sun, Xingxing Yan, Zhiye Wang and Xiuren Zhang, date: 04/05/2021, view: 201, Q&A: 0

[Abstract]

The majority of cellular proteins are degraded by the 26S proteasome in eukaryotes. However, intrinsically disordered proteins (IDPs), which contain large portions of unstructured regions and are inherently unstable, are degraded via the ubiquitin-independent 20S proteasome. Emerging evidence indicates that plant IDP homeostasis may also be

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Purification of Protein-complexes from the Cyanobacterium Synechocystis sp. PCC 6803 Using FLAG-affinity Chromatography

Authors: Minna M. Koskela, Petra Skotnicová, Éva Kiss and Roman Sobotka, date: 05/20/2020, view: 1999, Q&A: 0

[Abstract] Exploring the structure and function of protein complexes requires their isolation in the native state–a task that is made challenging when studying labile and/or low abundant complexes. The difficulties in preparing membrane-protein complexes are especially notorious. The cyanobacterium Synechocystis sp. PCC 6803 is a widely used model ...

Extraction and Purification of Laccases from Rice Stems

Authors: Chenna Swetha and P. V. Shivaprasad, date: 04/05/2019, view: 3394, Q&A: 0

[Abstract] Laccases are found in cell walls of plants in very low amounts. This protocol provides an efficient method to purify laccases from rice stems. The method involves three steps: 1) Isolation of total protein from rice stems using buffers with high salt concentration to extract protein from cell walls; 2) Purification of laccases using concanavalin-A ...

Isolation of Thylakoid Membranes from the Cyanobacterium Synechocystis sp. PCC 6803 and Analysis of Their Photosynthetic Pigment-protein Complexes by Clear Native-PAGE

Authors: Josef Komenda, Vendula Krynická and Tomas Zakar, date: 01/05/2019, view: 4271, Q&A: 0

[Abstract] Cyanobacteria represent a frequently used model organism for the study of oxygenic photosynthesis. They belong to prokaryotic microorganisms but their photosynthetic apparatus is quite similar to that found in algal and plant chloroplasts. The key players in light reactions of photosynthesis are Photosystem I and Photosystem II complexes (PSI and ...

Separation of Thylakoid Protein Complexes with Two-dimensional Native-PAGE

Authors: Marjaana Rantala, Virpi Paakkarinen and Eva-Mari Aro, date: 07/05/2018, view: 5984, Q&A: 0

[Abstract] The hierarchical composition and interactions of the labile thylakoid protein complexes can be assessed by sequential 2D-native gel-electrophoresis system. Mild non-ionic detergent digitonin is used to solubilize labile protein super-and megacomplexes, which are then separated with first-dimension blue native polyacrylamide gel electrophoresis ...

Investigating Localization of Chimeric Transporter Proteins within Chloroplasts of Arabidopsis thaliana

Authors: Susumu Uehara, Yasuko Ito-Inaba and Takehito Inaba, date: 02/05/2018, view: 6297, Q&A: 0

[Abstract] In this protocol, we describe a method to design chimeric proteins for specific targeting to the inner envelope membrane (IEM) of Arabidopsis chloroplasts and the confirmation of their localization by biochemical analysis. Specific targeting to the chloroplast IEM can be achieved by fusing the protein of interest with a transit peptide ...

Detection of Protein S-nitrosothiols (SNOs) in Plant Samples on Diaminofluorescein (DAF) Gels

Authors: Marta Rodríguez-Ruiz, Paulo T. Mioto, José M. Palma and Francisco J. Corpas , date: 09/20/2017, view: 5090, Q&A: 0

[Abstract] In plant cells, the analysis of protein S-nitrosothiols (SNOs) under physiological and adverse stress conditions is essential to understand the mechanisms of Nitric oxide (NO)-based signaling. We adapted a previously reported protocol for detecting protein SNOs in animal systems (King et al., 2005) for plant samples. Briefly, ...

Protocol for Enrichment of the Membrane Proteome of Mature Tomato Pollen

Authors: Puneet Paul, Palak Chaturvedi, Anida Mesihovic, Arindam Ghatak, Wolfram Weckwerth and Enrico Schleiff, date: 06/05/2017, view: 5664, Q&A: 0

[Abstract] We established and elaborated on a method to enrich the membrane proteome of mature pollen from economically relevant crop using the example of Solanum lycopersicum (tomato). To isolate the pollen protein fraction enriched in membrane proteins, a high salt concentration (750 mM of sodium chloride) was used. The membrane protein-enriched ...

Protein Isolation from Plasma Membrane, Digestion and Processing for Strong Cation Exchange Fractionation

Authors: Ning Yang, Bing Han and Tai Wang, date: 05/20/2017, view: 7834, Q&A: 0

[Abstract] Plasma membrane (PM) proteins play crucial roles in diverse biological processes. But their low abundance, alkalinity and hydrophobicity make their isolation a difficult task. This protocol describes an efficient method for PM proteins isolation, digestion and fractionation so that they can be well prepared for mass spectrometry analysis.

Rapid Isolation of Total Protein from Arabidopsis Pollen

Authors: Ming Chang and Shanjin Huang, date: 04/20/2017, view: 7667, Q&A: 0

[Abstract] Arabidopsis pollen is an excellent system for answering important biological questions about the establishment and maintenance of cellular polarity and polar cell growth, because these processes are amenable to the genetic and genomic approaches that are readily available in Arabidopsis. Given that proteins are the direct ...

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