An Improved Method for PAGE-based Detection of Phosphorylated Protein in Yeast
一种基于PAGE的检测酵母磷酸化蛋白的改进技术
发布: 2012年06月20日第2卷第12期 DOI: 10.21769/BioProtoc.210 浏览次数: 13288
PDF 
Q&A
Abstract
One dimensional polyacrylamide gel electrophoresis has been successfully used to detect protein phosphorylation. This method is very simple and highly reproducible. Hyperhosphorylated proteins usually migrate slowlier than dephosphorylated proteins. However, not all phosphorylated proteins can be readily detected, due to sub-optimal sample preparation and electrophoresis conditions. Here, an improved method is described that can detect phosphorylation of yeast proteins ranging from 15 kD to 200 kD. The improvement in gel electrophoresis should also be applicable to mammalian culture cells.
Keywords: Phosphorylation (磷酸化)
Yeast (酵母)
Mobility shift (迁移)
Acrylamide gel (丙烯酰胺凝胶)
Western blot (免疫印迹 (参照中文category))
Materials and Reagents
- Tris base (C4H11NO3) (Thermo Fisher Scientific, catalog number: 77-86-1 )
- NaCl (Thermo Fisher Scientific, catalog number: 7647-14-5 )
- EDTA (Na2EDTA•2H2O) (Sigma-Aldrich, catalog number: ED2SS )
- Triton X-100 (Thermo Fisher Scientific, catalog number: 9002-93-1 )
- Phenylmethanesulfonyl fluoride (PMSF) (C7H7FO2S) (Sigma-Aldrich, catalog number: P7626 )
- Complete protease inhibitor cocktail (F. Hoffmann-La Roche, catalog number: 04693159001 )
- PhosSTOP tablet (F. Hoffmann-La Roche, catalog number: 04906837001 )
- SDS [CH3 (CH2)11OSO3Na] (Sigma-Aldrich, catalog number: L3771 )
- Glycerol (Thermo Fisher Scientific, catalog number: 56-81-5 )
- β-mercaptoethanol (Sigma-Aldrich, catalog number: M7154 )
- Bromophenol blue (Sigma-Aldrich, catalog number: B0126 )
- Synthetic complete (SC) medium
- Rapamycin
- Methanol
- 5x SDS loading buffer (see Recipes)
- Yeast cell lysis buffer (see Recipes)
Equipment
- Standard bench-top centrifuge
- Sterilized toothpick
- Incubator
- Shaker
- 1.5 ml eppendorf tubes
- 25G needle
- Western blot equipment
- Refrigerator
Procedure
文章信息
版权信息
© 2012 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Wei, Y. (2012). An Improved Method for PAGE-based Detection of Phosphorylated Protein in Yeast. Bio-protocol 2(12): e210. DOI: 10.21769/BioProtoc.210.
Download Citation in RIS Format
分类
生物化学 > 蛋白质 > 修饰
微生物学 > 微生物生物化学 > 蛋白质 > 修饰
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。
提问指南
+ 问题描述
写下详细的问题描述，包括所有有助于他人回答您问题的信息（例如实验过程、条件和相关图像等）。
发布问题
