Non-Reducing SDS PAGE: A Method to Analyze Disulfide-Linked Multimeric Protein Complexes
Published: April 30, 2023
Abstract
Source: Rotoli, S. M. et al., Combining Non-reducing SDS-PAGE Analysis and Chemical Crosslinking to Detect Multimeric Complexes Stabilized by Disulfide Linkages in Mammalian Cells in Culture. J. Vis. Exp. (2019).
This video describes the technique of separating the protein samples by non-reducing SDS-PAGE to analyze the multimeric protein complexes. This technique retains the multimer subunits of a protein held by the disulfide bonds which can later be analyzed by western blotting.
Protocol
1. Blocking Free Cysteine Residues Using Iodoacetamide Grow U-2 OS cells in a 6 cm2 dish to 50%–60% confluency in minimum essential medium with high glucose containing 10% fetal bovine serum and 1% sodium pyruvate at 37 °C in 5% CO2. Make a fresh stock of 10 mM iodoacetamide just prior to use, then discard any unused reagent. Add 0.1 mM final concentration iodoacetamide directly to the cell culture media. Gently rock the dish at …
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The authors have nothing to disclose.
Materials
| 16% precast TGX gels | ThermoFisher | Xp00160 | |
| Bromophenol Blue | |||
| BSA | Cell signaling | 99985 | |
| Cell lysis buffer | Cell signaling | 9803 | |
| Centrifuge | Eppendorf | 5415D | |
| DMEM | Gibco | 11330-032 | |
| EDTA | Sigma Aldrich | M101 | |
| Electrophoresis apparatus | Invitrogen | A25977 | |
| Fetal bovine serum | Gibco | 1932693 | |
| Formaldehyde | ThermoFisher | 28908 | |
| Glass-teflon homogenizer | |||
| Glycerol | Sigma Aldrich | 65516 | |
| Glycine | RPI | 636050 | |
| Hepes | Sigma Aldrich | H0527 | |
| Iodoacetamide | ThermoFisher | 90034 | |
| PBS | Sigma Aldrich | P3813 | |
| Power supply | Biorad | 200120 | |
| Prestained marker | ThermoFisher | 26619 | |
| SDS | Biorad | 161-0302 | |
| Sonicator | Branson | 450 | |
| Tris Base | RPI | T60040 | |
| Tris Buffered Saline | Sigma Aldrich | SRE0031 | with Tween 20, pH 7.5 |
| Tris-Glycine running buffer | VWR | J61006 | |
| Triton X-100 | Sigma Aldrich | T8787 | |
| U-2 OS | ATCC | HTB-96 |
