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非还原 SDS-PAGE 分析与化学交联相结合, 检测培养基细胞中的多物质配合物, 以确认培养中的多物质配合
JoVE Journal
Developmental Biology
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JoVE Journal Developmental Biology
Combining Non-reducing SDS-PAGE Analysis and Chemical Crosslinking to Detect Multimeric Complexes Stabilized by Disulfide Linkages in Mammalian Cells in Culture
DOI:

09:37 min

May 02, 2019

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Chapters

  • 00:04Title
  • 00:34Harvesting Cells After Blocking Free Cysteine Residues Using Iodoacetamide
  • 02:02Extraction of Protein and Sample Preparation
  • 03:13In Vivo Formaldehyde Cross-linking of Endogenous Proteins in U-2 OS Cells
  • 04:30Fractionation of Nuclei
  • 06:11Extraction of Protein and Sample Preparation
  • 06:51SDS-PAGE Analysis
  • 07:53Results: Analyzing Multimeric Complexes Using a Two-step Method of Non-reducing SDS-PAGE Analysis and Formaldehyde Cross-linking
  • 09:04Conclusion

Summary

Automatic Translation

长期以来, 人们一直知道二硫化的联系可以稳定许多蛋白质的结构。通过非还原 SDS-PAGE 分析, 是分析通过这些联系稳定的多聚体复合物的一种简单方法。本文通过对人骨骨肉瘤细胞系 U-2 OS 的 dUTPase 核等形态的分析, 说明了该方法的应用。

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