Combining Non-reducing SDS-PAGE Analysis and Chemical Crosslinking to Detect Multimeric Complexes Stabilized by Disulfide Linkages in Mammalian Cells in Culture
Harvesting Cells After Blocking Free Cysteine Residues Using Iodoacetamide
2:02
Extraction of Protein and Sample Preparation
3:13
In Vivo Formaldehyde Cross-linking of Endogenous Proteins in U-2 OS Cells
4:30
Fractionation of Nuclei
6:11
Extraction of Protein and Sample Preparation
6:51
SDS-PAGE Analysis
7:53
Results: Analyzing Multimeric Complexes Using a Two-step Method of Non-reducing SDS-PAGE Analysis and Formaldehyde Cross-linking
9:04
Conclusion
Transcript
It has been well established that the structures of many proteins are stabilized in covalent disulfide linkages. In recent work this bond has been classified as a post-translational modification. Thus, it's important to be able to identify cystein
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Disulfide linkages have long been known to stabilize the structure of many proteins. A simple method to analyze multimeric complexes stabilized by these linkages is through non-reducing SDS-PAGE analysis. Here, this method is illustrated by analyzing the nuclear isoform of dUTPase from the human bone osteosarcoma cell line U-2 OS.