A Phosphopeptide Purification Protocol for the Moss Physcomitrella paten
小立碗藓磷肽的纯化方法
发布: 2015年07月20日第5卷第14期 DOI: 10.21769/BioProtoc.1527 浏览次数: 9722
评审: Valentine V TrotterArsalan Daudi
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参见作者原研究论文
The authors used this protocol in:
May 2014
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Abstract
Protein phosphorylation is one of the most common post-translational modifications in eukaryotic cells and plays a critical role in a vast array of cellular processes. Efficient methods of protein extraction and phosphopeptide purification are required to ensure the detection of high quality of proteins. In our hands, phenol extraction of proteins and TiO2 chromatography enrich phosphorylated peptides more efficiently than other methods in the moss Physcomitrlla patens (P. patens).
Materials and Reagents
- Seven-day old protonemata of the moss P. patens
- Liquid nitrogen
- Tris-HCl (pH 7.5)-saturated phenol (AMRESCO, catalog number: K168 )
- Ammonium acetate (Sigma-Aldrich, catalog number: A1542 )
- Methanol (Sigma-Aldrich, catalog number: 34860 )
- DTT (Promega Corporation, catalog number: V3155 )
- Acetone (Sigma-Aldrich, catalog number: 650501 )
- Iodoacetamide (Sigma-Aldrich, catalog number: I6125 )
- Ammonium bicarbonate (pH 8.5) (Sigma-Aldrich, catalog number: 40867 )
- Trypsin (Promega Corporation, catalog number: V5111 )
- Sucrose (Sigma-Aldrich, catalog number: S5390 )
- Tris (Sigma-Aldrich, catalog number: T1378 )
- EDTA (Sigma-Aldrich, catalog number: E6635 )
- 1,4-dithiothreitol (DTT) (Promega Corporation, catalog number: V3155)
- Protease inhibitors cocktail (Sigma-Aldrich, catalog number: P9599 )
- Phosphatase inhibitors cocktail 2 (Sigma-Aldrich, catalog number: P0044 )
- NH4OH (Sigma-Aldrich, catalog number: 320145 )
- Urea (Sigma-Aldrich, catalog number: U6504 )
- CHAPS (Sigma-Aldrich, catalog number: C9426 )
- Acetonitrile (ACN) (Sigma-Aldrich, catalog number: 34851 )
- Trifluoroacetic acid (TFA) (Sigma-Aldrich, catalog number: 302031 )
- Glutamic acid (Sigma-Aldrich, catalog number: G0355000 )
- Protein extraction buffer (see Recipes)
- Protein resuspension buffer (see Recipes)
- Loading buffer (see Recipes)
- Washing buffer I (see Recipes)
- Washing buffer II (see Recipes)
- Elution buffer (see Recipes)
Equipment
- Porcelain Mortar and Pestle (90 mm)
- Tubes and tube holder (2 ml)
- Titanium dioxide micro-columns (320 µm x 50 mm) (Column Technology, Freemont)
- Centrifuge
- Vortex
- Freeze dryer (Christ, model: Alpha 1-4 LSC )
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Wang, X. and He, Y. (2015). A Phosphopeptide Purification Protocol for the Moss Physcomitrella paten. Bio-protocol 5(14): e1527. DOI: 10.21769/BioProtoc.1527.
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分类
植物科学 > 植物生物化学 > 蛋白质 > 修饰
植物科学 > 植物生物化学 > 蛋白质 > 标记
系统生物学 > 蛋白质组学 > 磷酸化蛋白质组学
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