Non-Reducing SDS PAGE: A Method to Analyze Disulfide-Linked Multimeric Protein Complexes

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

1. 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.
2. Make a fresh stock of 10 mM iodoacetamide just prior to use, then discard any unused reagent.
3. Add 0.1 mM final concentration iodoacetamide directly to the cell culture media. Gently rock the dish at room temperature for 2 min.

2. Harvesting the Cells

1. Aspirate the media from the cells.....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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