从番茄果皮中分离液泡膜微囊

Christopher J. Snowden; Benjamin Thomas; Charles J. Baxter; J. Andrew C. Smith; Lee J. Sweetlove

doi:10.21769/BioProtoc.1686

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Peer-reviewed

Isolation of Tonoplast Vesicles from Tomato Fruit Pericarp

从番茄果皮中分离液泡膜微囊

CS Christopher J. Snowden

BT Benjamin Thomas

CB Charles J. Baxter

J. Andrew C. Smith

Lee J. Sweetlove email

发布: 2015年12月20日第5卷第24期 DOI: 10.21769/BioProtoc.1686 浏览次数: 9811

评审: Samik BhattacharyaAnonymous reviewer(s)

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The authors used this protocol in:

Cover of The Plant Journal, featuring study using the protocol.

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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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.