Extraction of Total Proteins from Rice Plant
从水稻植株中提取总蛋白
发布: 2014年11月05日第4卷第21期 DOI: 10.21769/BioProtoc.1277 浏览次数: 43635
评审: Feng LiCindy AstAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jan 2014
Abstract
This protocol provides an efficient method for preparation of high-quality proteins from rice leaves and grains. The method involves phenol extraction to separate proteins from the non-protein components such as polysaccharides, lipids and phenolic compounds that are commonly enriched in plant tissues. Following isolation, proteins are precipitated with ammonium acetate/methanol and then solubilized for proteomic analysis. As the protocol is simple, universal, and most importantly compatible with silver staining, it has been applied to our routine protein extraction from rice and many other plant tissues and it even works fine in animal tissues for the requirement of electrophoretic separation.
Keywords: Rice (水稻)
Protein extraction (蛋白质的提取)
Proteomics (蛋白质组学研究)
Two dimentional gel electrophresis (二维凝胶电泳)
Staining methods (染色方法)
Materials and Reagents
- Tainung 67
Note: TNG67, an elite japonica type rice variety, has been a leading variety in Taiwan for more than 30 years since it been released in 1978. - SA0420
Note: An aroma mutant, exhibits an agreeable taro-like flavor, was selected from the TNG67 mutation pool developed via sodium azide mutagenesis. - Liquid nitrogen
- Acetone (Merck KGaA, catalog number: 1.00012.4000 )
- NH4OAc (Merck KGaA, catalog number: 1.01116.1000 )
- DTT (Merck KGaA, catalog number: 1.11474.0001 )
- CHAPS (GE Healthcare, catalog number: 17-1314-01 )
- HCl (Merck KGaA, catalog number: 1.09063.1000 )
- 2-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
- 2-propanol (Merck KGaA, catalog number: 1.09634.2500 )
- Methanol (Merck KGaA, catalog number: 1.06007.4000 )
- NaCl (Merck KGaA, catalog number: 7710CN )
- NaOH (Merck KGaA, catalog number: 1.06469.1000 )
- KCl (Merck KGaA, catalog number: 1.04936.1000 )
- PMSF (Merck KGaA, catalog number: 52332 )
- Phenol (saturated, pH 6.6/7.9) (AMRESCO, catalog number: 0945 )
- PVPP (Merck KGaA, catalog number: 1.07302.0100 )
- Seasand (Merck KGaA, catalog number: 107711 )
- Sucrose (GERBU Biotechnik GmbH, catalog number: 1366 )
- Triton X-100 (GERBU Biotechnik GmbH, catalog number: 2000 )
- Tris-base (AMRESCO, catalog number: 0497 )
- Urea (AMRESCO, catalog number: 0378 )
- Miracloth (Calbiochem®, catalog number: 475855-1R )
- Extraction buffer (see Recipes)
- Precipitation buffer (see Recipes)
- 0.5 M EDTA (see Recipes)
- Lysis buffer (see Recipes)
- 40 mM PMSF (see Recipes)
Equipment
- Mortar (Φ 9 cm) and pestle (attached with Φ 12 cm mortar) (Figure 1)
Figure 1. Mortar and pestle - Pipetman (Gilson, model: p5000 , p1000 , p200 , p100 , p20 and p10 )
- High-Speed Refrigerated Centrifuges and No.46 Rotor (Hitachi, model: himac CR22F )
- 50 ml centrifuge tube (Thermo Fisher Scientific, Nalgene®, model: 3119-0050 )
- -20 °C refrigerator (Firstek Scientific, model: F-25 )
- SpeedVac system (Thermo Fisher Scientific, model: SPD speed Vac and UVS400 universal vacuum system )
- Ultrasonic machine (ULTRASONIC, model: LC130H )
- Chemical fume hood (CHUNG FU, model: HAP-090 )
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Lin, D. and Wang, C. (2014). Extraction of Total Proteins from Rice Plant. Bio-protocol 4(21): e1277. DOI: 10.21769/BioProtoc.1277.
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分类
植物科学 > 植物生物化学 > 蛋白质
生物化学 > 蛋白质 > 分离和纯化
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