通过非变性凝胶电泳法分离植物6-磷酸葡萄糖酸脱氢酶（6PGDH）同工型

Francisco J Corpas; Larisse de Freitas-Silva; Nuria García-Carbonero; Alba Contreras; Fátima Terán; Carmelo Ruíz-Torres; José M. Palma

doi:10.21769/BioProtoc.2399

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Separation of Plant 6-Phosphogluconate Dehydrogenase (6PGDH) Isoforms by Non-denaturing Gel Electrophoresis

通过非变性凝胶电泳法分离植物6-磷酸葡萄糖酸脱氢酶（6PGDH）同工型

Francisco J Corpas email

LF Larisse de Freitas-Silva

NG Nuria García-Carbonero

AC Alba Contreras

FT Fátima Terán

CR Carmelo Ruíz-Torres

José M. Palma

发布: 2017年07月20日第7卷第14期 DOI: 10.21769/BioProtoc.2399 浏览次数: 8781

评审: Amit DeyAnonymous reviewer(s)

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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, chloroplast, mitochondria and peroxisomes (Corpas et al., 1998; Krepinsky et al., 2001; Mateos et al., 2009; Fernández-Fernández and Corpas, 2016; Hölscher et al., 2016). Using Arabidopsis thaliana as plant model and sweet pepper (Capsicum annuum L.) fruits as a plant with agronomical interest, this protocol illustrates how to prepare the plant extracts for the separation of the potential 6PGDH isoforms by electrophoresis on 6% polyacrylamide non-denaturing gels. Thus, this method allows detecting three 6PGDH isoforms in Arabidopsis seedlings and two 6PGDH isoforms in sweet pepper fruits.

Keywords: NADPH (NADPH)

Non-denaturing polyacrylamide gel electrophoresis (非变性聚丙烯酰胺凝胶电泳)

Pentose phosphate pathway (磷酸戊糖途径)

6-Phosphogluconate dehydrogenase (6-磷酸葡萄糖酸脱氢酶)

Background

Non-denaturing gel electrophoresis is a powerful technique that allows separating native proteins. Their mobility depends of protein size, shape and net charge. In these analytical conditions, the protein preserves its activity and in combination with a specific staining method, it allows to separate the presence of potential isoforms. This approach has been widely used in the case of the family of superoxide dismutases. However, to our knowledge, there are not many papers that analyze the presence of different isoforms of 6PGDH activity in plant tissues (Corpas et al., 1998; Mateos et al., 2009). This straight method may be very useful for researchers working with plant 6PGDHs.

Materials and Reagents

10-cm-diameter Petri dishes
Arabidopsis thaliana ecotype Columbia seeds (originally obtained from NASC, Nottingham Arabidopsis Stock Center)
Sweet green pepper fruits were provided by Syngenta Seeds S.A. (El Ejido, Spain)
70% (v/v) ethanol
0.1% (w/v) SDS
Commercial Bleach
Murashige and Skoog medium (Sigma-Aldrich, catalog number: M5524 )
Phyto-agar (Duchefa Biochemie, catalog number: P1001 )
Sucrose (Sigma-Aldrich, catalog number: 84097 )
Bio-Rad Protein Assay (Bio-Rad Laboratories, catalog number: 5000006 )
Bovine serum albumin (BSA) Fraction V (Roche Diagnostics, catalog number: 10735078001 )
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 )
Dithiothreitol (DTT) (Roche Diagnostics, catalog number: 10708984001 )
30% acrylamide/Bis solution, 19:1 (Bio-Rad Laboratories, catalog number: 1610154 )
4x gel buffer
Glycine (AMRESCO, catalog number: 0167 )
Ammonium persulfate
TEMED
β-Nicotinamide adenine dinucleotide phosphate, sodium salt, hydrate (NADP) (Sigma-Aldrich, catalog number: N0505 )
Magnesium chloride, hexahydrate (MgCl₂·7H₂O) (EMD Millipore, catalog number: 442615 )
Nitroblue tetrazolium (NBT) (AMRESCO, catalog number: 0329-1G )
Phenazine methosulfate (PMS) (Sigma-Aldrich, catalog number: P9625 )
6-Phosphogluconic acid, trisodium salt (6PG) (Sigma-Aldrich, catalog number: P7877 )
Glacial acetic acid (Fisher Scientific)
Grinding buffer (see Recipes)
Non-denaturing polyacrylamide gel electrophoresis (PAGE) on 6% acrylamide gel (see Recipes)
Staining solution (see Recipes)
Stop solution (see Recipes)

Equipment

Plant Growth cabinet (Panasonic Biomedical, model: MLR-352-PE )
Porcelain mortar and pestle
Refrigerated centrifuge
Vertical Slab gels Electrophoresis System (Bio-Rad Laboratories, model: Mini-PROTEAN^® Electrophoresis Cell )

Software

ImageJ program (https://imagej.nih.gov/ij/)

Procedure

English

中文翻译

文章信息

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如何引用

Corpas, F. J., de Freitas-Silva, L., García-Carbonero, N., Contreras, A., Terán, F., Ruíz-Torres, C. and Palma, J. M. (2017). Separation of Plant 6-Phosphogluconate Dehydrogenase (6PGDH) Isoforms by Non-denaturing Gel Electrophoresis. Bio-protocol 7(14): e2399. DOI: 10.21769/BioProtoc.2399.