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Kombinerer ikke-reducerende SDS-PAGE analyse og kemisk crosslinking til Detect Multimeric komplekser stabiliseret af disulfid forbindelser i pattedyrceller i kultur
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

Disulfid forbindelser har længe været kendt for at stabilisere strukturen af mange proteiner. En enkel metode til at analysere multimeriske komplekser stabiliseret af disse forbindelser er gennem ikke-reducerende SDS-Page analyse. Her illustreres denne metode ved at analysere den nukleare isoformen af dutpase fra den menneskelige knogle osteosarkom cellelinje U-2 os.

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