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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Introduction

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 interaction1,2,3,4. 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....

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.......

Representative Results

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.......

Discussion

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 .......

Acknowledgements

We gratefully appreciate the efforts put forth by Dr. Jennifer Fischer for the purification of the dUTPase polyclonal antibody and Kerri Ciccaglione for all her efforts to help edit this manuscript. This research was partially supported by a grant from the New Jersey Health Foundation (Grant #PC 11-18).

....

Materials

NameCompanyCatalog NumberComments
16% precast TGX gelsThermoFisherXp00160
175 cm2 FlaskCell star658175
18COATCCCRL-1459
6 cm2 dishVWR10861-588
A549ATCCCCL-185
Amersham ECL detection kitGE16817200
Blot transfer apparatusBiorad153BR76789
BMESigma AldrichM3148
Bradford protein reagentBiorad5000006
Bromophenol Blue
BSACell signaling99985
Cell lysis bufferCell signaling9803
CentrifugeEppendor5415D
DMEMGibco11330-032
Drill
EDTASigma AldrichM101
Electrophoresis apparatusInvitrogenA25977
Extra thick western blotting paperThermoFisher88610
Fetal bovine serumGibco1932693
FormaldehydeThermoFisher28908
Glass-teflon homogenizer
GlycerolSigma Aldrich65516
GlycineRPI636050
Heat blockDenville10285-D
HepesSigma AldrichH0527
Hydrochloric acidVWR2018010431
IodoacetamideThermoFisher90034
KimwipeKimtech34155
MethanolPharmco339000000
Non-fat dry milkCell signaling99995
PBSSigma AldrichP3813
PMSFSigma Aldrich329-98-6
Posi-click tubeDenvilleC2170
Power supplyBiorad200120
Prestained markerThermoFisher26619
PVDF membraneBiorad162-0177
RockerReliable Scientific55
Saos2ATCCHTB-85
SDSBiorad161-0302
Secondary antibodyCell signaling70748
Small cell scraperTygonS-50HL class VI
Sodium chlorideRPIS23020
Sodium pyruvateGibco
SonicatorBranson450
Sponge pad for blottingInvitrogenE19051
Stir plateCorningPC353
SucroseSigma AldrichS-1888
Tris BaseRPIT60040
Tris Buffered Saline, with Tween 20, pH 7.5Sigma AldrichSRE0031
Tris-Glycine running bufferVWRJ61006
Triton X-100Sigma AldrichT8787
Tween 20Sigma AldrichP9416
U-2 OSATCCHTB-96
X-ray filmThermoFisher34090

References

  1. Klomsiri, C., Karplus, P. A., Poole, L. B. Cysteine-based redox switches in enzymes. Antioxidants and Redox Signaling. 14 (6), 1065-1077 (2011).
  2. Antelmann, H., Helmann, J. D. Thiol-based redox switches and gene regulation. Antioxi....

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Disulfide LinkagesMultimeric ComplexesNon reducing SDS PAGEChemical CrosslinkingMammalian CellsProtein StructureCysteine stabilized ComplexesIodoacetamideCell LysisBradford AssayLaemmli SDS PAGEFormaldehyde Crosslinking

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