在二氨基荧光素（DAF）凝胶上检测植物样品中的蛋白质S-亚硝基硫醇（SNO）

Marta Rodríguez-Ruiz; Paulo T. Mioto; José M. Palma; Francisco J. Corpas

doi:10.21769/BioProtoc.2559

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Peer-reviewed

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

在二氨基荧光素（DAF）凝胶上检测植物样品中的蛋白质S-亚硝基硫醇（SNO）

MR Marta Rodríguez-Ruiz

PM Paulo T. Mioto

José M. Palma

Francisco J. Corpas email

发布: 2017年09月20日第7卷第18期 DOI: 10.21769/BioProtoc.2559 浏览次数: 7535

评审: Swetha ReddyWenrong HeAnonymous reviewer(s)

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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, proteins from plant samples are separated via non-reducing SDS-PAGE, then the NO bound by S-nitrosylated proteins is released using UV light and, finally, the NO is detected using the fluorescent probe DAF-FM (Rodriguez-Ruiz et al., 2017). Thus, the approach presented here provides a relatively quick and economical procedure that can be used to compare protein SNOs content in plant samples and provide insight in NO-based signaling in plants.

Keywords: Nitric oxide (一氧化氮)

S-nitrosothiols (S-亚硝基硫醇)

S-nitrosation (S-亚硝化)

S-nitrosylation (S-亚硝基化)

Background

Nitric oxide (NO) is a free radical which can interact with a diverse array of biomolecules including proteins, lipids, and nucleic acids. In the case of proteins, one of the most relevant post-translational modifications (PTMs) is the covalent attachment of an NO group to the thiol (-SH) side chain of cysteine (Cys) present in peptides or proteins. This modification generates a family of NO-derived molecules called S-nitrosothiols (SNOs) which are important compounds in both animal and plant systems (Foster et al., 2003; Lindermayr and Durner, 2009; Astier et al., 2011; Broniowska and Hogg, 2012). Although this PTM is often designated as S-nitrosylation, the more appropriate term is S-nitrosation. It is difficult to detect, quantify and identify protein SNOs in plant systems. While there are several techniques to detect SNOs such as chemiluminescence, the biotin switch method, mass spectrometry, fluorescence detection, and antibody detection (against S-nitrosocysteine) (Kettenhofen et al., 2007; Foster, 2012; Devarie-Baez et al., 2013; Diers et al., 2014; Barroso et al., 2016; Mioto et al., 2017) many of these techniques require tedious sample preparation procedures that are time consuming and require sophisticated, expensive equipment.

Materials and Reagents

10-cm-diameter polystyrene Petri dishes (Fisher Scientific, catalog number: 12654785 )
Parafilm M All-Purpose Paraffin Wax Film (Bemis, catalog number: PM996 )
Sweet green pepper fruits were provided by Syngenta Seeds S.A. (El Ejido, Spain)
Note: This company grows pepper plants in experimental glass-covered greenhouses under optimal conditions of light, temperature and humidity.
Arabidopsis thaliana ecotype Columbia seeds (originally obtained from NASC, Nottingham Arabidopsis Stock Center)
Ethanol (Fisher Scientific, catalog number: 10517694 )
Commercial Bleach (20%)
Murashige and Skoog medium (Sigma-Aldrich, catalog number: M5524 )
Sucrose (Sigma-Aldrich, catalog number: 84097 )
Phyto-agar (Sigma-Aldrich, catalog number: P8169-100G )
Bio-Rad Protein Assay Dye Reagent (Bio-Rad Laboratories, catalog number: 5000006 )
Bovine serum albumin (BSA) Fraction V (Roche Diagnostics, Sigma-Aldrich, catalog number: 10735078001 )
4-20% Precast TGX Mini-Protean gel (Bio-Rad Laboratories, catalog number: 4561093 )
Ascorbate (AsA) (Sigma-Aldrich, catalog number: A7631-25G )
Copper(I) chloride (CuCl) (Sigma-Aldrich, catalog number: 651745-5G )
N-ethylmaleimide (NEM) (Sigma-Aldrich, catalog number: E3876-5G )
Dithiothreitol (DTT) (Roche Diagnostics, catalog number: 10708984001 )
Reduced glutathione (GSH) (Sigma-Aldrich, catalog number: G4251-5G )
β-Mercaptoethanol (ME) (Sigma-Aldrich, catalog number: M6250-10ML )
Tris (AMRESCO, catalog number: 0497 )
Ethylenediaminetetraacetic acid, disodium salt, dihydrate (Na₂-EDTA) (Sigma-Aldrich, catalog number: E5134 )
Triton X-100 (AMRESCO, catalog number: 0694 )
Glycerol (AMRESCO, catalog number: E520 )
Sodium dodecyl sulfate (SDS; electrophoresis grade)
Bromophenol blue (Sigma-Aldrich, catalog number: B0126-25G )
3-Amino,4-aminomethyl-2’,7’-difluorescein (DAF-FM) (Sigma-Aldrich, catalog number: D2196 )
Grinding buffer (see Recipes)
Sample treatment buffer (2x) (see Recipes)
Standard running buffer for SDS-PAGE containing 1 mM EDTA (see Recipes)
Gel staining solution (see Recipes)

Equipment

Set of Gilson micropipettes (Gilson, P10, P20 and P100)
Plant growth cabinet (Panasonic Biomedical, model: MLR-352-PE )
Porcelain mortar and pestle (VWR, catalog numbers: 410-0110 and 410-0120 , respectively)
Refrigerated centrifuge Hettich Mikro 220R (Hettich Lab Technology, model: Mikro 220 R , catalog number: 2205)
Vertical Slab gels Electrophoresis System (Bio-Rad Laboratories, model: Mini-PROTEAN^®, catalog number: 1658003EDU )
Standard UV-transilluminator (302-312 nm), used in molecular biology laboratory
Molecular Imager PharosFX system (Bio-Rad Laboratories, model: PharosFX^TM, catalog number: 1709460 )
Note: This product has been discontinued.
Evolution^TM201 UV-visible spectrophotometer (Thermo Fisher Scientific, Thermo Scientific^TM, model: Evolution^TM 201 , catalog number: 912A0890)

Software

ImageJ (free application available in https://imagej.net/)

Procedure

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Rodríguez-Ruiz, M., Mioto, P. T., Palma, J. M. and Corpas, F. J. (2017). Detection of Protein S-nitrosothiols (SNOs) in Plant Samples on Diaminofluorescein (DAF) Gels. Bio-protocol 7(18): e2559. DOI: 10.21769/BioProtoc.2559.