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Characterising Plant Deubiquitinases with in vitro Activity-based Labelling and Ubiquitin Chain Disassembly Assays

Authors: Michael J. Skelly and Steven H. Spoel, date: 05/05/2021, view: 380, Q&A: 0

[Abstract]

Post-translational modification of proteins by ubiquitin is an essential cellular signaling mechanism in all eukaryotes. Ubiquitin is removed from target proteins by a wide range of deubiquitinase (DUB) enzymes with different activities and substrate specificities. Understanding how DUBs function in vitro is a vital first step to uncovering their

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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: 445, 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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Non-radioactive Assay to Determine Product Profile of Short-chain Isoprenyl Diphosphate Synthases

Authors: Avanish Rai and Dinesh A. Nagegowda, date: 01/05/2021, view: 841, Q&A: 0

[Abstract] Isoprenoids represent the largest class of metabolites with amazing diversities in structure and function. They are involved in protecting plants against pathogens or herbivores or involved in attracting pollinators. Isoprenoids are derived from geranyl diphosphate (GPP; C₁₀), farnesyl diphosphate (FPP; C₁₅), geranylgeranyl ...

The ATPase Activity of Escherichia coli Expressed AAA⁺-ATPase Protein

Authors: Amita Kaundal, Vemanna S. Ramu and Kirankumar S. Mysore, date: 08/05/2020, view: 1617, Q&A: 0

[Abstract] ATPases are the enzymes that breakdown ATP to ADP and release inorganic phosphate (Pi). Here we provide a detailed protocol to determine the ATPase activity of a recombinant AAA⁺-ATPase protein (GENERAL CONTROL NON-REPRESSIBLE-4 [GCN4]) by spectrophotometric absorption at 360 nm to measure the accumulated inorganic phosphate. In ...

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: 2297, 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 ...

Simple Method to Determine Protein Redox State in Arabidopsis thaliana

Authors: Keisuke Yoshida and Toru Hisabori, date: 06/05/2019, view: 3872, Q&A: 0

[Abstract] Thiol-based redox regulation is a posttranslational protein modification that plays a key role in many biological aspects. To understand its regulatory functions, we need a method to directly assess protein redox state in vivo. Here we present a simple procedure to determine protein redox state in a model plant Arabidopsis thaliana ...

Extraction and Purification of Laccases from Rice Stems

Authors: Chenna Swetha and P. V. Shivaprasad, date: 04/05/2019, view: 3579, 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 ...

Detection of Disulfides in Protein Extracts of Arabidopsis thaliana Using Monobromobimane (mBB)

Authors: Shin‐nosuke Hashida and Maki Kawai-Yamada, date: 03/05/2019, view: 3835, Q&A: 0

[Abstract] Thiol-disulfide exchange is a key posttranslational modification, determining the folding process of intra- and inter-protein structures. Thiols can be detected by colorimetric reagents, which are stoichiometrically reduced by free thiols, and by fluorescent adducts, showing fluorescence only after thioester formation. We adapted a simple ...

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: 4492, 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 ...

Enzymatic Assays and Enzyme Histochemistry of Tuta absoluta Feeding on Tomato Leaves

Authors: Rim Hamza, José P. Beltrán and Luis A. Cañas, date: 09/05/2018, view: 4482, Q&A: 0

[Abstract] Enzymes play a key role in insect-plant relationships. For a better understanding of these interactions, we analyzed Tuta absoluta digestive enzymes. Here, we describe a detailed protocol for the detection of trypsin and papain-like enzymes in Tuta absoluta larvae by enzyme histochemistry. This assay uses frozen and unfixed ...

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