Plasma Membrane Preparation from Lilium davidii and Oryza sativa Mature and Germinated Pollen
兰州百合和亚洲水稻成熟和萌发花粉的浆膜制备
发布: 2017年05月20日第7卷第10期 DOI: 10.21769/BioProtoc.2297 浏览次数: 9075
评审: Samik BhattacharyaCindy AstAnonymous reviewer(s)
PDF 
Q&A
参见作者原研究论文
The authors used this protocol in:
Dec 2010
Advertisement
Abstract
Pollen germination is an excellent process to study cell polarity establishment. During this process, the tip-growing pollen tube will start elongating. The plasma membrane as the selectively permeable barrier that separates the inner and outer cell environment plays crucial roles in this process. This protocol described an efficient aqueous polymer two-phase system followed by alkaline solution washing to prepare Lilium davidii or Oryza sativa plasma membrane with high purity.
Keywords: Mature pollen grains (成熟花粉粒)
Germinated pollen grains (萌发花粉粒)
Plasma membran (浆膜)
Aqueous polymer two-phase system (水性聚合物两相体系)
Alkaline solution (碱性溶液)
Background
Pollen plasma membrane contains various proteins that are vital for pollen tube growth and fertilization, such as receptor-like kinases (Wang et al., 2016) and ion channels (Hamilton et al., 2015). Isolating pure plasma membrane (PM) is the premise for the comprehensive PM proteome analysis. There are mainly four methods for PM preparation: differential centrifugation, density gradient centrifugation, preparative free-flow electrophoresis and the aqueous polymer two-phase system. Normally, differential centrifugation is often combined with density gradient centrifugation together to separate the subcellular components according to their size, shape and density. This technique is rapid, but due to the organelle density’s overlap, the resultant PM yield and purity are low (Schindler and Nothwang, 2006). Both free-flow electrophoresis and the aqueous polymer two-phase system separate membrane vesicles according to their surface properties. These two methods can enrich PM pure enough for proteomic analysis (Alexandersson et al., 2007). However, the instrument for the free-flow electrophoresis is complicated to operate (Sandelius et al., 1986). On the contrast, the aqueous polymer two-phase system can be performed easily and rapidly with centrifugation, making this method more convenient for PM preparation. PM enriched by the aqueous polymer two-phase system present in the form of vesicles which contain some cytoplasm contaminations (Alexandersson et al., 2008). Treatment with alkaline solution (100 mM Na2CO3, pH 11.5) can open these vesicles into sheets to release the contaminations (Fujiki et al., 1982).
Materials and Reagents
- 1,000 μl pipette tips (Corning, Axygen®, catalog number: T-1000-B )
- 20 x 10 cm envelope
- 50 ml tube (Corning, catalog number: 430829 )
- 60 x 15 mm Petri dish (Corning, catalog number: 430196 )
- 150 x 25 mm Petri dish (Corning, catalog number: 430599 )
- Gauze
- 10 ml tube (Biosharp, catalog number: BS-100-M )
- 100 μm cell strainer (Corning, Falcon®, catalog number: 352360 )
- 2 ml microtube (SARSTEDT, catalog number: 72.694.005 )
- 4 ml ultracentrifuge tube (Beckman Coulter, catalog number: 355603 )
- 26.3 ml ultracentrifuge tube (Beckman Coulter, catalog number: 355654 )
- Lily mature pollen grains harvest according to Han et al. (2010)
- Rice mature pollen grains harvest according to Dai et al. (2007)
- Boric acid (H3BO3) (Sigma-Aldrich, catalog number: B9645 )
- Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
- Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C2661 )
Note: This product has been discontinued. - Sucrose (Sigma-Aldrich, catalog number: S7903 )
- Calcium nitrate tetrahydrate, Ca(NO3)2·4H2O (Sigma-Aldrich, catalog number: C1396 )
- Thiamine hydrochloride (VB1) (Sigma-Aldrich, catalog number: T4625 )
- Poly (ethylene glycol), average Mn 4,000 (PEG 4000) (Sigma-Aldrich, catalog number: 81240 )
- 3-(N-Morpholino)propanesulfonic acid (MOPS) (Sigma-Aldrich, catalog number: M1254 )
- Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
- DL-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
- Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
- cOmplete, EDTA-free protease inhibitor cocktail tablets (Roche Diagnostics, catalog number: 04693132001 )
- L-ascorbic acid (VC) (Sigma-Aldrich, catalog number: A7506 )
- Poly (vinylpolypyrrolidone) (PVPP) (Sigma-Aldrich, catalog number: 77627 )
- Potassium phosphate tribasic (K3PO4) (Sigma-Aldrich, catalog number: P5629 )
- Polyethylene glycol, average mol wt 3,350 (PEG 3350) (Sigma-Aldrich, catalog number: P4338 )
- Dextran T-500 (Pharmacia, catalog number: 17-0320-01 )
- Sodium carbonate (Na2CO3) (Sigma-Aldrich, catalog number: S7795 )
- Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A1933 )
- Lily pollen germination medium (see Recipes)
- Rice pollen germination medium (see Recipes)
- Homogenate buffer (see Recipes)
- Plasma membrane isolation buffer (see Recipes)
- Aqueous polymer two-phase system (see Recipes)
- Dilution buffer (see Recipes)
- Washing buffer (see Recipes)
Equipment
- Pipette (Gilson, model: P1000N )
- Vortex
- Balance
- Centrifuge (Beckman Coulter, model: J2-HS )
- Homogenizer (MP Biomedicals, model: FastPrep®-24 )
- Ultra-centrifuge (Beckman Coulter, model: OptimalTM L-80XP )
- Microscope with 5x and 10x objective (Carl Zeiss, model: Axio Imager 1 )
Software
- ZEN lite software (2012, blue edition)
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Yang, N., Han, B., Liu, L., Yang, H. and Wang, T. (2017). Plasma Membrane Preparation from Lilium davidii and Oryza sativa Mature and Germinated Pollen. Bio-protocol 7(10): e2297. DOI: 10.21769/BioProtoc.2297.
Download Citation in RIS Format
分类
植物科学 > 植物细胞生物学 > 细胞器分离
植物科学 > 植物细胞生物学 > 细胞分离
细胞生物学 > 细胞器分离 > 细胞膜
您对这篇实验方法有问题吗？
在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。
发布问题
