Extraction of Intracellular and Cell Wall Proteins from Leaves and Roots of Harsh Hakea
从粗糙哈克木属植物叶和根中提取胞内和细胞壁蛋白质
发布: 2015年12月05日第5卷第23期 DOI: 10.21769/BioProtoc.1678 浏览次数: 9906
评审: Arsalan DaudiSollapura J. VishwanathKabin Xie
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参见作者原研究论文
The authors used this protocol in:
Nov 2014
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Abstract
Plant proteins can be targeted to intracellular (i.e., cytosol, vacuole, organelles etc.) or extracellular (i.e., cell walls, apoplast) compartments. Dual targeting is a key mechanism with important implications for plant metabolism, growth, development and defense etc. Harsh Hakea (Hakea prostrata R.Br.) is a perennial species and member of the Proteaceae family that thrives on extremely phosphate impoverished soils of southwestern Australia. Harsh Hakea is not a common model organism, but has been widely developed for physiological and molecular/biochemical studies of the endogenous adaptations of an ‘extremophile’ plant species to abiotic stress, including low phosphorus tolerance. Tissues of Harsh Hakea contain large amounts of compounds (e.g., phenolics) that interfere with the extraction of soluble proteins. We previously optimised extraction of intracellular proteins from Harsh Hakea proteoid roots to improve soluble protein yield by at least 10-fold (Shane et al., 2013). Here, we describe the protocol for extraction and separation of intracellular from ‘loosely bound’ cell-wall proteins in Harsh Hakea.
Materials and Reagents
- 2 ml microcentrifuge tubes (Fisher Scientific, catalog number: 05-408-138 )
- Dialysis tubing (12-14 kDa MWCO) (Spectra/Por, Fisher Scientific, catalog number: 08-667B )
- 25 mm syringe filters (0.45 μm) (Sarstedt, catalog number: 83.1826 )
- Harsh Hakea leaves or roots
- Dewar (MVE 20/15) with liquid N2, and ice bucket
- Imidazole (Bioshop, catalog number: IMID508 )
- Triton X-100 (Bioshop, catalog number: TRX777 )
- Glycerol (Bioshop, catalog number: GLY002 )
- Magnesium chloride (Bioshop, catalog number: MAG508 )
- Polyethylene glycol 8000 (Bioshop, catalog number: PEG800 )
- Phenylmethylsulfonyl fluoride (PMSF, G-Biosciences, catalog number: 786-0555 )
- Thiourea (Sigma, catalog number: T-7875 )
- Poly (vinylpolypyrrolidone) (PVPP, Sigma-Aldrich, catalog number: P-6755 )
- Poly (vinylpyrrolidone) (Bioshop, catalog number: PVP504 )
- Intracellular extraction buffer (see Recipes)
- Resuspension buffer (see Recipes)
- Cell wall extraction buffer (see Recipes)
- Dialysis buffer (see Recipes)
Equipment
- ZebaTM spin desalting columns 7K MWCO (0.5 ml) (Fisher Scientific, catalog number: 89882 )
- Porcelain mortar (70 mm inner diameter) and pestle
- Plastic pellet pestle (Kimble-Chase Kontes, Fisher Scientific, catalog number: K749521-0590 )
- Macrosep® Advance Centrifugal Concentrators (10 kDa MWCO) (Pall Corporation, catalog number: MAP010C37 )
- Microcentrifuge (Beckman Coulter, model: microfuge 22R )
- PT-3100 Polytron Homogenizer (Kinematica, model: PT-3100)
- Magnetic stirrer
- 4 °C cold room
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Shane, M. W. and Plaxton, W. (2015). Extraction of Intracellular and Cell Wall Proteins from Leaves and Roots of Harsh Hakea. Bio-protocol 5(23): e1678. DOI: 10.21769/BioProtoc.1678.
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分类
植物科学 > 植物生物化学 > 蛋白质 > 分离和纯化
植物科学 > 植物细胞生物学 > 组织分析
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