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2D Diagonal Redox SDS-PAGE of Proteins

蛋白质二维双向还原电泳技术

CS Christian Schwarz
JN Joerg Nickelsen

发布: 2013年06月05日第3卷第11期 DOI: 10.21769/BioProtoc.781 浏览次数: 15992

评审: Tie Liu

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Abstract

2D diagonal redox SDS-PAGE of proteins is used to detect intramolecular or intermolecular disulfide bridges using Chlamydomonas in this example (Stroeher and Dietz, 2008; Schwarz et al., 2012). Both dimensions consist of a conventional SDS-PAGE, except that the sample buffer for the first dimension lacks a reducing agent. Intermolecular disulfide bridges increase the apparent molecular weight of a protein in the first dimension, whereas intramolecular bridges decrease the apparent weight of the protein.

Keywords: 2d Diagonal SDS PAGE (二维对角线电泳) search Redox (氧化还原) search Disulfide bridge (二硫桥) search

Materials and Reagents

  1. All material for a conventional Laemmli SDS-PAGE
  2. Non-reducing Laemmli sample buffer
  3. KCl (Applichem, catalog number: A1039 )
  4. Triton X-100 (Applichem, catalog number: A1388 )
  5. DTT (Applichem, catalog number: A1101 )
  6. SDS (SERVA Electrophoresis GmbH, catalog number: 20765 )
  7. Acrylamide 40% 37.5:1 (SERVA Electrophoresis GmbH, catalog number: 10681 )
  8. Ammoniumperoxodisulfate (Carl Roth, catalog number: 9592 )
  9. TEMED (Carl Roth, catalog number: 2367 )
  10. Beta-mercaptoethanol (Carl Roth, catalog number: 4227 )
  11. Iodoacetamide (Applichem, catalog number: A1666 )
  12. Tris (Applichem, catalog number: A1379 )
  13. Na2EDTA (Carl Roth, catalog number: 8043 )
  14. Tricine (Carl Roth, catalog number: 6977 )
  15. Glycine (SERVA Electrophoresis GmbH, catalog number: 23390 )
  16. Agarose (SERVA Electrophoresis GmbH, catalog number: 11380 )
  17. Bromophenol blue (Carl Roth, catalog number: T116 )
  18. Filter paper (Munktell, catalog number: 2.519.580600N )
  19. Page Ruler unstained protein ladder (Thermo Fisher Scientific, catalog number: 26614 )
  20. 2x Cell lysis buffer (see Recipes)
  21. 5x non-reducing Laemmli sample buffer (see Recipes)
  22. 5x reducing Laemmli sample buffer (see Recipes)
  23. Laemmli electrophoresis buffer (see Recipes)

Equipment

  1. All equipment for a conventional Laemmli SDS-PAGE

Procedure

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文章信息

版权信息

© 2013 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Schwarz, C. and Nickelsen, J. (2013). 2D Diagonal Redox SDS-PAGE of Proteins. Bio-protocol 3(11): e781. DOI: 10.21769/BioProtoc.781.

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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 > 电泳

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