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Subcellular Fractionation of Hela Cells for Lysosome Enrichment Using a Continuous Percoll-density Gradient

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Simple Method to Determine Protein Redox State in Arabidopsis thaliana

Authors: Keisuke Yoshida and Toru Hisabori, date: 06/05/2019, view: 1106, 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: 1063, 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: 1144, 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: 1800, 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: 1912, 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: 2982, 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: 3771, 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: 1929, 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 ...

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: 3480, 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 ...

In vitro RNA-dependent RNA Polymerase Assay Using Arabidopsis RDR6

Authors: Kyungmin Baeg, Yukihide Tomari and Hiro-oki Iwakawa, date: 01/05/2018, view: 3514, 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 ...

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