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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.
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.
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 technique used in many laboratories, where the results can be obtained and interpreted quickly, easily, and with minimal costs, and is advantageous over other techniques used to identify disulfide linkages such as mass spectrometry6,7 and circular dichroism8.
One important step in determining if this method is an appropriate technique to aid in a study is to thoroughly examine the primary sequence of the protein of interest to insure there are cysteine residue(s) present. Another helpful step is to research any previous crystal structures published or use a bioinformatics application to explore the three dimensional structure of the protein of interest to visualize where the cysteine residue(s) may be located. If the residue(s) is present on the outside surface it may be a better candidate to form a disulfide linkage rather than a cysteine residue buried on the inside of the structure. However, it is important to note that proteins may undergo structural changes upon substrate interactions or protein-protein interactions allowing these residues to then become exposed to the environment as well.
Identified multimeric complexes can then be verified with chemical cross-linking using formaldehyde. Formaldehyde is an ideal cross-linker for this verification technique due to the high cell permeability and short cross-linking span of ~2-3 Å, ensuring detection of specific protein-protein interactions9,10. Here, this method is illustrated by analyzing the nuclear isoform of dUTPase from the human bone osteosarcoma cell line U-2 OS11. However, this protocol can be adapted for other cell lines, tissues and organisms.
1. Blocking Free Cysteine Residues Using Iodoacetamide
2. Harvesting the Cells
3. Extraction of Protein
4. Sample Preparation
5. In Vivo Formaldehyde Cross-linking of Endogenous Proteins in U-2 OS Cells
6. Fractionation of Nuclei
7. Extraction of Protein
8. Sample Preparation
9. SDS-PAGE Analysis
10. Western Blot
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...
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 ...
The authors declare that they have no conflicts of interest with the contents of this manuscript.
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).
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 |
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