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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: 770, 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: 1240, 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: 2986, 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: 2760, 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: 3115, 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: 3614, 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: 3629, 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 ...

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

Authors: Marjaana Rantala, Virpi Paakkarinen and Eva-Mari Aro, date: 07/05/2018, view: 5031, 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 ...

In vitro Nitrate Reductase Activity Assay from Arabidopsis Crude Extracts

Authors: Joo Yong Kim and Hak Soo Seo, date: 04/05/2018, view: 6248, Q&A: 0

[Abstract] Nitrate reductase (NR) reduces the major plant nitrogen source, NO₃^-, into NO₂^-. NR activity can be measured by its final product, nitrite through its absorbance under optimized condition. Here, we present a detailed protocol for measuring relative enzyme activity of NR from Arabidopsis crude ...

Histone Deubiquitination Assay in Nicotiana benthamiana

Authors: Shujing Liu and Lars Hennig, date: 03/05/2018, view: 3239, Q&A: 0

[Abstract] Histone modifications are a group of post-translational modifications on histones which can alter chromatin structure and affect gene expression. Histone ubiquitination is a histone modification found in particular on histone H2A and H2B. Histone ubiquitination can be reversed by ubiquitin-specific proteases (UBP). Here, we describe an in vivo ...

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