Isolation of Tonoplast Vesicles from Tomato Fruit Pericarp
从番茄果皮中分离液泡膜微囊
发布: 2015年12月20日第5卷第24期 DOI: 10.21769/BioProtoc.1686 浏览次数: 10478
评审: Samik BhattacharyaAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Mar 2015
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Abstract
This protocol describes the isolation of tonoplast vesicles from tomato fruit. The vesicles isolated using this procedure are of sufficiently high purity for downstream proteomic analysis whilst remaining transport competent for functional assays. The methodology was used to study the transport of amino acids during tomato fruit ripening (Snowden et al., 2015) and based on the procedure used by Betty and Smith (Bettey and Smith, 1993). Such vesicles may be useful in further studies into the dynamic transfer of metabolites across the tonoplast for storage and metabolism during tomato fruit development.
Materials and Reagents
- Ultra centrifuge tubes, 32 ml capacity, thick wall, polycarbonate (Beckman Coulter, catalog number: 355631 )
- 3 ml pastettes
- Solanum lycopersicum Lam. cv. M82 or cv. Micro-Tom
- Bovine serum albumin (Fraction V) (Sigma-Aldrich, catalog number: A7906 )
- D-Mannitol (Sigma-Aldrich, catalog number: M9647 )
- Magnesium sulfate heptahydrate (Thermo Fisher Scientific, catalog number: 10256840 )
- Ethylenediaminetetraacetic acid disodium salt dihydrate (Sigma-Aldrich, catalog number: E4884 )
- Polyvinylpyrrolidone average molecular weight 40,000 (Sigma-Aldrich, catalog number: PVP40 )
- 2-Amino-2-(hydroxymethyl)-1, 3-propanediol (Trizma® base) (Sigma-Aldrich, catalog number: T1503 )
- Butylated hydroxytoluene (Sigma-Aldrich, catalog number: W218405 )
- Potassium disulfite (K2S2O5) (Sigma-Aldrich, catalog number: P2522 )
- Phenylmethylsulfonyl fluouride (Sigma-Aldrich, catalog number: P7626 )
- Ethanol (Thermo Fisher Scientific, catalog number: 12468750 )
- (+)-Sodium L-ascorbate (Sigma-Aldrich, catalog number: A7631 )
- DL-Dithiothreitol (Sigma-Aldrich, catalog number: D0632 )
- Potassium hydroxide (Thermo Fisher Scientific, catalog number: 10366240 )
- Glycerol (Thermo Fisher Scientific, catalog number: 10296200 )
- BIS-TRIS propane (Sigma-Aldrich, catalog number: B6755 )
- Tricine (Sigma-Aldrich, catalog number: T0377 )
- Sucrose (Sigma-Aldrich, catalog number: S0389 )
- Muslin cloth (100 % cotton)
- 2-(N-morpholino)ethanesulfonic acid (MES) (Sigma-Aldrich, catalog number: M3671 )
- Extraction buffer (see Recipes)
- Resuspension buffer (see Recipes)
- Sucrose steps (see Recipes)
- Transport buffer (see Recipes)
Equipment
- Scalpel Handle Swan Morton No.4 (Philip Harris, catalog number: B8R00181 )
- Scalpel blades Swan Morton No. 24 (Thermo Fisher Scientific, catalog number: 11712734 )
- Blender (For example a Waring 38BL41 with a 40 oz container)
- Ultra centrifuge capable of 100,000 x g with swing out rotor (e.g. SW28 Ti)
- Soft paint brush (Sable No.5 or equivalent)
- 1,000 µl micropipette
Procedure
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文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Snowden, C. J., Thomas, B., Baxter, C. J., Smith, J. A. C. and Sweetlove, L. J. (2015). Isolation of Tonoplast Vesicles from Tomato Fruit Pericarp. Bio-protocol 5(24): e1686. DOI: 10.21769/BioProtoc.1686.
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分类
植物科学 > 植物生物化学 > 蛋白质 > 分离和纯化
生物化学 > 蛋白质 > 分离和纯化
细胞生物学 > 细胞器分离 > 细胞膜
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