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Generation of Giant Unilamellar Vesicles (GUVs) Using Polyacrylamide Gels

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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: 501, 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: 1256, 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 ...

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: 4037, 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 ...

In vitro Nitrate Reductase Activity Assay from Arabidopsis Crude Extracts

Authors: Joo Yong Kim and Hak Soo Seo, date: 04/05/2018, view: 6861, 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 ...

In vitro RNA-dependent RNA Polymerase Assay Using Arabidopsis RDR6

Authors: Kyungmin Baeg, Yukihide Tomari and Hiro-oki Iwakawa, date: 01/05/2018, view: 6465, Q&A: 0

[Abstract] RNA-dependent RNA polymerases (RdRPs) in eukaryotes convert single-stranded RNAs into double-stranded RNAs, thereby amplifying small interfering RNAs that play crucial roles in the regulation of development, maintenance of genome integrity and antiviral immunity. Here, we describe a method of in vitro RdRP assay using recombinant ...

Determination of H⁺-ATPase Activity in Arabidopsis Guard Cell Protoplasts through H⁺-pumping Measurement and H⁺-ATPase Quantification

Authors: Shota Yamauchi and Ken-ichiro Shimazaki, date: 12/20/2017, view: 5777, Q&A: 0

[Abstract] The opening of stomata in plants in response to blue light is driven by the plasma membrane H⁺-ATPase in guard cells. To evaluate the activation of the H⁺-ATPase in vivo, we can use H⁺-pumping by guard cells in response to blue light and fusicoccin. To do this, it is required to prepare a large amount of ...

Isolation and Detection of the Chlorophyll Catabolite Hydroxylating Activity from Capsicum annuum Chromoplasts

Authors: Mareike Hauenstein and Stefan Hörtensteiner, date: 09/20/2017, view: 5646, 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: 6158, 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

Authors: Francisco J Corpas, Larisse de Freitas-Silva, Nuria García-Carbonero, Alba Contreras, Fátima Terán, Carmelo Ruíz-Torres and José M. Palma, date: 07/20/2017, view: 6079, Q&A: 1

[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: 7900, 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 ...

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