/
/
Kombinerer ikke-reducerende SDS-PAGE analyse og kemisk crosslinking til Detect Multimeric komplekser stabiliseret af disulfid forbindelser i pattedyrceller i kultur
A subscription to JoVE is required to view this content. Sign in or start your free trial.
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
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.
