Combining Non-reducing SDS-PAGE Analysis and Chemical Crosslinking to Detect Multimeric Complexes Stabilized by Disulfide Linkages in Mammalian Cells in Culture

Riepilogo

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.

Abstract

The structures of many proteins are stabilized through covalent disulfide linkages. In recent work, this bond has also been classified as a post-translational modification. Thus, it is important to be able to study this modification in living cells. A simple method to analyze these cysteine-stabilized multimeric complexes is through a two-step method of non-reducing SDS-PAGE analysis and formaldehyde cross-linking. This two-step method is advantageous as the first step to uncovering multimeric complexes stabilized by disulfide linkages due to its technical ease and low cost of operation. Here, the human bone osteosarcoma cell line U-2 OS is used to illustrate this method by specifically analyzing the nuclear isoform of dUTPase.

Introduzione

Disulfide linkages have long been known to stabilize the structure of many proteins. In recent work, this bond has also been classified as a reversible post-translational modification, acting as a cysteine-based "redox switch" allowing for the modulation of protein function, location and interaction^1,2,3,4. Thus, it is important to be able to study this modification. A simple method to analyze these cysteine-stabilized multimeric complexes is through non-reducing SDS-PAGE analysis⁵. SDS-PAGE analysis is a tech....

Protocollo

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.
2. Wash the cells with 5 mL of ....

Risultati

Nuclear dUTPase forms an intermolecular disulfide linkage forming a stable dimer configuration through the interaction of two cysteine residues positioned at the third amino acid of each monomeric protein¹¹. This is demonstrated in Figure 1A,B. To ensure this disulfide linkage was not a nonspecific interaction due to migration abnormalities in the non-reduced environment,t the inclusion of a proper control was essentia.......

Discussione

The method outlined here gives a straight-forward protocol for the analysis of multimeric complexes stabilized through disulfide linkages. This protocol can easily be adapted to other cell culture lines, tissues and organisms allowing for a broad range of applications.

An important step in this procedure is to ensure the disulfide linkages are not a consequence of the extraction procedure. Any free cysteine residues can be blocked using iodoacetamide¹³. This alkylating .......

Materiali

Name	Company	Catalog Number	Comments
16% precast TGX gels	ThermoFisher	Xp00160
175 cm2 Flask	Cell star	658175
18CO	ATCC	CRL-1459
6 cm2 dish	VWR	10861-588
A549	ATCC	CCL-185
Amersham ECL detection kit	GE	16817200
Blot transfer apparatus	Biorad	153BR76789
BME	Sigma Aldrich	M3148
Bradford protein reagent	Biorad	5000006
Bromophenol Blue
BSA	Cell signaling	99985
Cell lysis buffer	Cell signaling	9803
Centrifuge	Eppendor	5415D
DMEM	Gibco	11330-032
Drill
EDTA	Sigma Aldrich	M101
Electrophoresis apparatus	Invitrogen	A25977
Extra thick western blotting paper	ThermoFisher	88610
Fetal bovine serum	Gibco	1932693
Formaldehyde	ThermoFisher	28908
Glass-teflon homogenizer
Glycerol	Sigma Aldrich	65516
Glycine	RPI	636050
Heat block	Denville	10285-D
Hepes	Sigma Aldrich	H0527
Hydrochloric acid	VWR	2018010431
Iodoacetamide	ThermoFisher	90034
Kimwipe	Kimtech	34155
Methanol	Pharmco	339000000
Non-fat dry milk	Cell signaling	99995
PBS	Sigma Aldrich	P3813
PMSF	Sigma Aldrich	329-98-6
Posi-click tube	Denville	C2170
Power supply	Biorad	200120
Prestained marker	ThermoFisher	26619
PVDF membrane	Biorad	162-0177
Rocker	Reliable Scientific	55
Saos2	ATCC	HTB-85
SDS	Biorad	161-0302
Secondary antibody	Cell signaling	70748
Small cell scraper	Tygon	S-50HL class VI
Sodium chloride	RPI	S23020
Sodium pyruvate	Gibco
Sonicator	Branson	450
Sponge pad for blotting	Invitrogen	E19051
Stir plate	Corning	PC353
Sucrose	Sigma Aldrich	S-1888
Tris Base	RPI	T60040
Tris Buffered Saline, with Tween 20, pH 7.5	Sigma Aldrich	SRE0031
Tris-Glycine running buffer	VWR	J61006
Triton X-100	Sigma Aldrich	T8787
Tween 20	Sigma Aldrich	P9416
U-2 OS	ATCC	HTB-96
X-ray film	ThermoFisher	34090