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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

Yuehua Wei Yuehua Wei

发布: 2012年06月20日第2卷第12期 DOI: 10.21769/BioProtoc.210 浏览次数: 13481

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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) search Yeast (酵母) search Mobility shift (迁移) search Acrylamide gel (丙烯酰胺凝胶) search Western blot (免疫印迹 (参照中文category)) search

Materials and Reagents

  1. Tris base (C4H11NO3) (Thermo Fisher Scientific, catalog number: 77-86-1 )
  2. NaCl (Thermo Fisher Scientific, catalog number: 7647-14-5 )
  3. EDTA (Na2EDTA•2H2O) (Sigma-Aldrich, catalog number: ED2SS )
  4. Triton X-100 (Thermo Fisher Scientific, catalog number: 9002-93-1 )
  5. Phenylmethanesulfonyl fluoride (PMSF) (C7H7FO2S) (Sigma-Aldrich, catalog number: P7626 )
  6. Complete protease inhibitor cocktail (F. Hoffmann-La Roche, catalog number: 04693159001 )
  7. PhosSTOP tablet (F. Hoffmann-La Roche, catalog number: 04906837001 )
  8. SDS [CH3 (CH2)11OSO3Na] (Sigma-Aldrich, catalog number: L3771 )
  9. Glycerol (Thermo Fisher Scientific, catalog number: 56-81-5 )
  10. β-mercaptoethanol (Sigma-Aldrich, catalog number: M7154 )
  11. Bromophenol blue (Sigma-Aldrich, catalog number: B0126 )
  12. Synthetic complete (SC) medium
  13. Rapamycin
  14. Methanol
  15. 5x SDS loading buffer (see Recipes)
  16. Yeast cell lysis buffer (see Recipes)

Equipment

  1. Standard bench-top centrifuge
  2. Sterilized toothpick
  3. Incubator
  4. Shaker
  5. 1.5 ml eppendorf tubes
  6. 25G needle
  7. Western blot equipment
  8. Refrigerator

Procedure

English
中文翻译

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.

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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

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