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).

1. Blocking Free Cysteine Residues Using Iodoacetamide
<ol>
	<li>Grow U-2 OS cells in a 6 cm2 dish to 50%&#8211;60% con&#64258;uency in minimum essential medium with high glucose containing 10% fetal bovine serum and 1% sodium pyruvate at 37 &#176;C in 5% CO2.</li>
	<li>Make a fresh stock of 10 mM iodoacetamide just prior to use, then discard any unused reagent.</li>
	<li>Add 0.1 mM final concentration iodoacetamide directly to the cell culture media. Gently rock the dish at room temperature for 2 min.</li>
</ol>
2. Harvesting the Cells
<ol>
	<li>Aspirate the media from the cells.</li>
	<li>Wash the cells with 5 mL of cold phosphate-buffered saline (PBS) three times. Aspirate the final PBS wash solution then add 1 mL of cold PBS.</li>
	<li>Scrape the cells off the bottom of the dish using a cell scraper. Using a 1 mL pipette draw up the PBS and cell suspension and dispense all the liquid into a 1.5 mL microcentrifuge tube.</li>
	<li>Spin the cells at 7,500 x g for three min at 4 &#176;C. Aspirate the PBS, leaving the cell pellet behind. The cell pellet can be stored at -80 &#176;C until processing</li>
</ol>
3. Extraction of Protein
<ol>
	<li>Prepare 100 &#181;L of a 1x lysis buffer diluted in ddH2O (See Table of Materials). Add phenylmethylsulfonyl fluoride (PMSF) immediately before use to a 1 mM final concentration.</li>
	<li>Add 50 &#181;L of the 1x lysis buffer directly to cell pellet and suspend. Incubate this on ice for 5 min.</li>
	<li>Sonicate for 8 s using the constant pulse mode at 40% (see Table of Materials for sonicator; adjust as necessary for different sonicators), keeping the extract on ice.</li>
	<li>Spin the extract at 4 &#176;C for 5 min at 16,000 x g. The supernatant is the soluble protein fraction. Bradford analysis can be performed to determine protein concentration if desired.</li>
</ol>
4. Sample Preparation
<ol>
	<li>Take 10 &#181;L of the soluble protein extract and add 10 &#181;L of a 1x Laemmli SDS-sample buffer (4% SDS, 20% glycerol, 0.004% bromophenol blue, and 0.125 M Tris-HCl, pH 6.8). Do not add any reducing reagents.</li>
	<li>Keep samples on ice if SDS PAGE analysis will be performed that day; for long term storage, -20 &#176;C is appropriate. Right before running the gel, heat the sample for 5 min at 85 &#176;C.</li>
</ol>
5. In Vivo Formaldehyde Cross-linking of Endogenous Proteins in U-2 OS Cells
<ol>
	<li>Grow U-2 OS cells in a 175 cm2 flask to 70%&#8211;80% con&#64258;uency (see section 1).</li>
	<li>Perform the formaldehyde crosslinking reaction
	<ol>
		<li>In a fume hood, aliquot a 37% formaldehyde solution purchased from commercial sources. Add the formaldehyde fixative directly to the medium to a &#64257;nal concentration of 1% and incubate at room temperature with gentle agitation for 15 min.</li>
		<li>To quench the reaction, add 1.25 M glycine to a final concentration of 0.125 M and incubate at room temperature with gentle agitation on a rocker for 5 min.</li>
	</ol>
	</li>
	<li>Wash the cells with 5 mL of cold PBS three times. Aspirate the final PBS wash solution and add 10 mL of PBS. Scrape the cells off the bottom of the flask using a cell scraper.</li>
	<li>Using a 10 mL pipette, draw up the PBS and cell suspension and dispense all of the liquid into a 15 mL conical centrifuge tube. Spin the cells at 500 x g for 2 min at 4 &#176;C. Aspirate the PBS, leaving the cell pellet behind.</li>
</ol>
6. Fractionation of Nuclei
<ol>
	<li>Prepare 10 mL of homogenization buffer: 0.25 M sucrose, 1 mM EDTA, 10 mM HEPES, and 0.5% BSA at pH 7.4. Add PMSF immediately before use to a 1 mM final concentration and 3mL of nuclei suspension buffer (0.1% Triton X-100 in PBS).</li>
	<li>Add 5 mL homogenization buffer directly to the cell pellet and suspend completely. Centrifuge the suspension at 500 x g for 2 min at 4 &#176;C then discard the supernatant.</li>
	<li>Suspend the pellet in 5 mL of homogenization buffer. Use a tight-fitting glass-Teflon homogenizer, dounce homogenize cells at 10 strokes/500 rpm.</li>
	<li>Centrifuge the suspension at 1,500 x g for 10 min at 4 &#176;C, then discard the supernatant. 
	NOTE: Use the supernatant for isolation of mitochondria by centrifuging at 10,000 x g for 10 min.</li>
	<li>Suspend the pellet in 1 mL nuclei suspension buffer and incubate on ice for 10 min.</li>
	<li>Centrifuge at 600 x g for 10 min. Discard the supernatant.</li>
	<li>Suspend the pellet in 1 mL nuclei suspension buffer and centrifuge again. Discard the supernatant. The final pellet will be isolated nuclei.</li>
</ol>
7. Extraction of Protein
<ol>
	<li>Repeat section 4, except adding 25 &#181;L of the 1x lysis buffer directly to the cell pellet then suspending.</li>
</ol>
8. Sample Preparation
<ol>
	<li>Prepare two samples for SDS-PAGE by taking 10 &#181;L each of the soluble protein extract and add 10 &#181;L of 2x Laemmli SDS-sample buffer and 1 &#181;L of 2-Mercaptoethanol (BME).</li>
	<li>Heat one sample for 5 min at 37 &#176;C and the second sample for 15 min at 98 &#176;C to reverse the formaldehyde cross link.</li>
</ol>
9. SDS-PAGE Analysis
<ol>
	<li>Prepare 1 L of 1x Tris-glycine running buffer (25 mM Tris, 192 mM Glycine, 0.1% (w/v) SDS).</li>
	<li>Set up the SDS-PAGE running apparatus. 
	NOTE: This protocol uses a 16% precast TGX SDS-Page. Of note, any percentage gel can be used.
	<ol>
		<li>Per the manufacturer protocol, open the package the gel is stored in and remove the cassette.</li>
		<li>Remove the comb that is lining the wells and the tape from the bottom of the cassette.</li>
		<li>Place the gel into the running apparatus.</li>
		<li>Fill the chamber with the 1x running buffer until the wells are submerged in liquid. Using a plastic pipet, rinse out the wells with the running buffer.</li>
	</ol>
	</li>
	<li>Load the samples on the gel along with 10 &#181;L prestained standard marker.</li>
	<li>Run the gel at 200 V until the dye front is approximately 1 cm from the bottom of the gel.</li>
</ol>
10. Western Blot
<ol>
	<li>Prepare 1 L of 1x transfer buffer (25 mM Tris, 192 mM Glycine, 20% methanol) and store at 4 &#176;C until use.</li>
	<li>Carefully remove the gel and open the cassette. Using a razor, carefully cut and discard the stacking gel. Pick up the gel using one corner and place it into a tray with transfer buffer, allowing it to rock gently for 5 min.</li>
	<li>While the gel is rocking, place an ice block (stored in -20 &#176;C) into the transfer tank and add the transfer buffer to &#190; full.</li>
	<li>Wet the polyvinylidene fluoride (PVDF) membrane by soaking it in 100% methanol for 30 s.</li>
	<li>Once the 5 min have passed, prepare to set up the transfer in a tray with transfer buffer. The transfer buffer should be enough to completely submerge the blot. Set up the blot transfer as follows: at bottom, the bottom of the cassette holder (black); then a thick sponge, extra thick blotting paper, the polyacrylamide gel, a PVDF membrane, extra thick blotting paper, and a thick sponge; then finally the top of the cassette holder (white) at top.</li>
	<li>Lock the cassette and place the unit into the transfer tank with the PVDF membrane towards the positive anode and the gel towards the negative. Top off the unit with transfer buffer until your transfer is completely submerged.</li>
	<li>Place the unit on top of a stir plate. Add a stir bar to the unit and begin stirring at 125 rpm.</li>
	<li>Run at 100 V for 60 min.</li>
	<li>While the transfer is running, prepare a solution of 5% powdered milk dissolved in Tris-buffered saline, with Tween-20, pH 7.5 (TBST).</li>
	<li>Dismantle the unit and remove the membrane, noting which side was in contact with the gel; ensure this side stays up in the tray for the remaining steps.</li>
	<li>Soak the membrane for 2 min in Tris-buffered saline (TBS) followed by blocking the membrane by incubating in 5 mL of 5% milk-TBST solution for 30 min at room temperature.</li>
	<li>Replace the milk with a fresh 4 mL of 5% milk-TBST solution and add the primary antibody at the proper dilution (in this case, our dilution is 1:2,000 for the dUTPase antibody) and incubate over night with gentle rocking at 4 &#176;C.</li>
	<li>The following day, remove the blot from the 4 &#176;C rocker to a room temperature rocker, discarding the primary antibody solution.</li>
	<li>Do three quick washes with TBST, using enough liquid to completely submerge the blot, followed by 3 washes, 5 min each, rocking slowly.</li>
	<li>Following the last wash, add 5 mL of the 5% milk-TBST solution and rock the blot for 15 min then discard.</li>
	<li>Add secondary antibody diluted in 5% milk-TBST at the desired concentration. (In this case, 1:5,000 dilution of goat anti-rabbit diluted in 5 mL of 5% milk-TBST solution)</li>
	<li>Incubate at room temperature, rocking for 1 h, then discard the solution.</li>
	<li>Do three quick washes with TBST followed by 3 washes, 5 min each, rocking slowly, then discard the final wash and add 5 mL of TBS.</li>
	<li>Prepare a 1:1 mixture of an ECL chemiluminescent detection solutions (1 mL of each reagent for a final volume of 2 mL) and add this directly to the blot incubating for 1 min.</li>
	<li>Remove the membrane using tweezers and dab the corner with a laboratory wipe, removing any excess solution. Place the membrane in shrink wrap and place the protein side up in an x-ray cassette.</li>
	<li>Expose the membrane to x-ray film. The time of exposure will vary.</li>
</ol>

<ol>
	<li>Klomsiri, C., Karplus, P. A., Poole, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cysteine-based+redox+switches+in+enzymes.">Cysteine-based redox switches in enzymes.</a> Antioxidants and Redox Signaling. 14 (6), 1065-1077 (2011).</li><li>Antelmann, H., Helmann, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiol-based+redox+switches+and+gene+regulation.">Thiol-based redox switches and gene regulation.</a> Antioxidants and Redox Signaling. 14 (6), 1049-1063 (2011).</li><li>Brandes, N., Schmitt, S., Jakob, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiol-based+redox+switches+in+eukaryotic+proteins.">Thiol-based redox switches in eukaryotic proteins.</a> Antioxidants and Redox Signaling. 11 (5), 997-1014 (2009).</li><li>Groitl, B., Jakob, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiol-based+redox+switches.">Thiol-based redox switches.</a> Biochimica et Biophysica Acta. 1844 (8), 1335-1343 (2014).</li><li>Stern, B. D., Wilson, M., Jagus, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+nonreducing+SDS-PAGE+for+monitoring+renaturation+of+recombinant+protein+synthesis+initiation+factor,+eIF-4+alpha.">Use of nonreducing SDS-PAGE for monitoring renaturation of recombinant protein synthesis initiation factor, eIF-4 alpha.</a> Protein Expression and Purification. 4 (4), 320-327 (1993).</li><li>Gorman, J. J., Wallis, T. P., Pitt, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+disulfide+bond+determination+by+mass+spectrometry.">Protein disulfide bond determination by mass spectrometry.</a> Mass Spectrometry Reviews. 21 (3), 183-216 (2002).</li><li>Li, X., Yang, X., Hoang, V., Liu, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Protein+Disulfide+Linkages+by+MS+In-Source+Dissociation+Comparing+to+CID+and+ETD+Tandem+MS.">Characterization of Protein Disulfide Linkages by MS In-Source Dissociation Comparing to CID and ETD Tandem MS.</a> Journal of the American Society for Mass Spectrometry. , (2018).</li><li>Kelly, S. M., Price, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+circular+dichroism+in+the+investigation+of+protein+structure+and+function.">The use of circular dichroism in the investigation of protein structure and function.</a> Current Protein and Peptide Science. 1 (4), 349-384 (2000).</li><li>Klockenbusch, C., Kast, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+formaldehyde+cross-linking+for+protein+interaction+analysis+of+non-tagged+integrin+beta1.">Optimization of formaldehyde cross-linking for protein interaction analysis of non-tagged integrin beta1.</a> Journal of Biomedicine and Biotechnology. 2010, 927585 (2010).</li><li>Gavrilov, A., Razin, S. V., Cavalli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+formaldehyde+cross-linking:+it+is+time+for+black+box+analysis.">In vivo formaldehyde cross-linking: it is time for black box analysis.</a> Briefings in Functional Genomics. 14 (2), 163-165 (2015).</li><li>Rotoli, S. M., Jones, J. L., Caradonna, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cysteine+residues+contribute+to+the+dimerization+and+enzymatic+activity+of+human+nuclear+dUTP+nucleotidohydrolase+(nDut).">Cysteine residues contribute to the dimerization and enzymatic activity of human nuclear dUTP nucleotidohydrolase (nDut).</a> Protein Science. , (2018).</li><li>Caradonna, S., Muller-Weeks, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nature+of+enzymes+involved+in+uracil-DNA+repair:+isoform+characteristics+of+proteins+responsible+for+nuclear+and+mitochondrial+genomic+integrity.">The nature of enzymes involved in uracil-DNA repair: isoform characteristics of proteins responsible for nuclear and mitochondrial genomic integrity.</a> Current Protein and Peptide Science. 2 (4), 335-347 (2001).</li><li>Macpherson, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noxious+compounds+activate+TRPA1+ion+channels+through+covalent+modification+of+cysteines.">Noxious compounds activate TRPA1 ion channels through covalent modification of cysteines.</a> Nature. 445 (7127), 541-545 (2007).</li><li>Carey, M. F., Peterson, C. L., Smale, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+immunoprecipitation+(ChIP).">Chromatin immunoprecipitation (ChIP).</a> Cold Spring Harbor Protocols. 2009 (9), (2009).</li></ol>

The authors declare that they have no conflicts of interest with the contents of this manuscript.

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 agent will bind covalently to cysteine residues though their thiol group, blocking the formation of new disulfide bonds. However, if the disulfide bond is present at the time of treatment this agent will not disrupt it. Optimization of the iodoacetamide can be done using a variation of concentration and time. However, over exposure to this reagent will cause cell death.
An additional step to this protocol that may be helpful is the optimization of the formaldehyde cross-linking. A variation of the percent of formaldehyde can be used as well as the time of cross-linking9,14. It is important to note that as the percent of formaldehyde as well as time increases, so do the chances of forming non-specific interactions. Downstream confirmation of the multimeric complex is also necessary. A useful technique to determine the molecular weight and identity, if the complex is a heteromultimeric, is mass spectrometry. Also, site directed mutagenesis can be a suitable technique to determine the cysteine residues responsible for the disulfide linkage.
Lastly, this two-step method of non-reduced SDS-PAGE analysis and formaldehyde cross-linking verification is beneficial due to its technical ease and low cost. It can be a first step in uncovering multimeric complexes stabilized by disulfide linkages.

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 &#34;redox switch&#34; 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 &#197;, 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.

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

combining non-reducing sds-page analysis and chemical crosslinking to detect multimeric complexes stabilized by disulfide linkages in mammalian cells in culture

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.

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 essential. Of note, nuclear dUTPase is one of four isoforms present in humans. Three of the four isoforms have a unique amino-terminal domain while sharing a common catalytic core11,12. As seen on the western blot, the monomeric confirmation of dUTPase in human cells at the time of harvesting is a combination of at least three of the isoforms of dUTPase (the mitochondria isoform, the nuclear isoform, and a truncated version notated at M24), all of which are recognized by our polyclonal antibody. The nuclear isoform is the only isoform that contains a cysteine residue in its unique amino terminal domain. Due to the nuclear isoform being only a small percentage of the monomeric state of the proteins seen on the western blot, it was exposed longer to demonstrate the dimeric state of that isoform.
The mitochondrial isoform lacks the cysteine residue present in nuclear isoform and was used as a control for this intermolecular disulfide linkage. As seen in Figure 1C, isolation of mitochondria followed by a western blot analysis demonstrated that this isoform under non-reducing conditions did not form a disulfide linkage and migrated to the predicted molecular weight for the monomeric protein.
To confirm this complex can form a multimeric complex, formaldehyde cross-linking was performed. Isolated nuclei were subjected to 1% formaldehyde treatment followed by denaturing SDS-PAGE/western blot analysis under reducing conditions. As demonstrated in Figure 2, the dimerization was visualized. When the cross-link was reversed by incubating the sample at 95 °C for 15 min, the complex was destabilized and could be visualized in its monomeric state.
<img alt="Gel electrophoresis result showing protein bands with and without BME treatment across different samples." class="xfigimg" src="/files/ftp_upload/59483/59483fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Demonstration of intermolecular disulfide bond formation in the nuclear dUTPase protein. (A) A western blot analysis of total cell extracts (TCE) in the absence of the reducing agent, beta-mercaptoethanol (BME), demonstrates a multimeric complex formation in asynchronous populations of U-2 OS, Saos2, A549, and 18CO as indicated by the black box. (B) This complex disappears with the addition of BME in all four cell lines examined, indicating the presence of a disulfide linkage. (C) A western blot analysis of TCE and purified mitochondrial extracts (Mito) derived from U-2 OS cells, ±BME, shows no multimeric complex formation in the -BME sample. The lower panels in panels A, B, and C demonstrate exposure to X-ray film for 10 s, showing the monomeric state of the three isoforms of dUTPase. The upper panels in A and B were exposed for 1 min, while the upper panel in C was exposed for 2 min. Equivalent amounts of protein were applied to each lane. Blots were probed with a polyclonal specific antibody against the conserved carboxyl-terminal domain of dUTPase. This figure has been modified from Rotoli et al.11 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59483/59483fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Electrophoresis results of formaldehyde-treated mitochondrial and nuclear protein fractions." class="xfigimg" src="/files/ftp_upload/59483/59483fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Formaldehyde cross-linking of nuclear dUTPase demonstrate multimeric complex formation. U-2 OS cells were incubated with 1% formaldehyde for 15 min. Nuclei (N) were isolated then analyzed by western blot using a specific polyclonal antibody against the conserved carboxyl-terminal domain of dUTPase (+ formaldehyde). To reverse the formaldehyde cross-links, extracts derived from the nuclear preparations were mixed with SDS-PAGE buffer then heated to 98 °C for 15 min in the presence of BME (+formaldehyde, 98 °C for 15 min). The observed heterogeneity (i.e. doublet bands of nDut) seen with the preparations remain to be explained, but may be due to anomalous migration due to the formaldehyde treatment. The lower panel is exposed to X-ray film for 10 s, while the upper panel is exposed to X-ray film for 2 min. This figure has been modified from Rotoli et al.11 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59483/59483fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Combining Non-reducing SDS-PAGE Analysis and Chemical Crosslinking to Detect Multimeric Complexes Stabilized by Disulfide Linkages in Mammalian Cells in Cultu

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

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 ...

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Nanyang Technological University

Research

JoVE Journal

Developmental Biology

10.0K Views.  Rowan University, School of Osteopathic Medicine and Graduate School of Biomedical Sciences. Het is goed vastgesteld dat de structuren van veel eiwitten zijn gestabiliseerd in covalente disulfide koppelingen. In recent werk is deze band geclassificeerd als een post-translationele wijziging. Het is dus belangrijk om cysteïne gestabiliseerde multimercomplexen in levende cellen te kunnen identificeren met behulp van een methode die snel en eenvoudig is.Het belangrijkste voordeel van deze techniek is dat de resultaten snel, gemakkelijk en met minimale kosten kunnen worden verkreg...

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

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

Chemistry

Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.

This article describes a simple and rapid protocol to evaluate the oligomeric state of the dynamin-like GTPase MxA protein from lysates of human cells using a combination of non-denaturing PAGE with western blot analysis.

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral ...

Biochemistry

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by networks of interacting protein complexes, which are often difficult to investigate because their binding is non-covalent and easily perturbed by purification techniques. This work describes a method of stabilizing and separating native protein complexes from unmodified tissue using two-dimensional polyacrylamide gel electrophoresis. Tissue lysate is loaded onto a non-denaturing blue-native polyacrylamide gel, then an electric current is applied until the protein migrates a short distance into the gel. The gel strip containing the migrated protein is then excised and incubated with the amine-reactive cross-linking reagent dithiobis(succinimidyl propionate), which covalently stabilizes protein complexes. The gel strip containing cross-linked complexes is then cast into a sodium dodecyl sulfate polyacrylamide gel, and the complexes are separated completely. The method relies on techniques and materials familiar to most molecular biologists, meaning it is inexpensive and easy to learn. While it is limited in its ability to adequately separate extremely large complexes, and has not been universally successful, the method was able to capture a wide variety of well-studied complexes, and is likely applicable to many systems of interest.

A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented.

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by ...

Synthesis and Structure Determination of &#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus highly important to avoid undesired disulfide bond formation during peptide synthesis, because it may result in a completely different peptide structure, and consequently altered bioactivity. However, the correct formation of multiple disulfide bonds in a peptide is difficult to obtain by using standard self-folding methods such as conventional buffer oxidation protocols, because several disulfide connectivities can be formed. This protocol represents an advanced strategy required for the targeted synthesis of multiple disulfide-bridged peptides which cannot be synthesized via buffer oxidation in high quality and quantity. The study demonstrates the application of a distinct protecting group strategy for the synthesis of all possible 3-disulfide-bonded peptide isomers of &#181;-conotoxin PIIIA in a targeted way. The peptides are prepared by Fmoc-based solid phase peptide synthesis using a protecting group strategy for defined disulfide bond formation. The respective pairs of cysteines are protected with trityl (Trt), acetamidomethyl (Acm), and tert-butyl (tBu) protecting groups to make sure that during every oxidation step only the required cysteines are deprotected and linked. In addition to the targeted synthesis, a combination of several analytical methods is used to clarify the correct folding and generation of the desired peptide structures. The comparison of the different 3-disulfide-bonded isomers indicates the importance of accurate determination and knowledge of the disulfide connectivity for the calculation of the three-dimensional structure and for interpretation of the biological activity of the peptide isomers. The analytical characterization includes the exact disulfide bond elucidation via tandem mass spectrometry (MS/MS) analysis which is performed with partially reduced and alkylated derivatives of the intact peptide isomer produced by an adapted protocol. Furthermore, the peptide structures are determined using 2D nuclear magnetic resonance (NMR) experiments and the knowledge obtained from MS/MS analysis.

Cysteine-rich peptides fold into distinct three-dimensional structures depending on their disulfide connectivity. Targeted synthesis of individual disulfide isomers is required when buffer oxidation does not lead to the desired disulfide connectivity. The protocol deals with the selective synthesis of 3-disulfide-bonded peptides and their structural analysis using NMR and MS/MS studies.

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus ...

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, and release of the complex components. Glycerol gradient centrifugation has been used for the separation of protein or RNP (RiboNucleoProtein) complexes for functional and structural studies. Here, we describe the utilization of Grafix (Gradient Fixation), which was first developed to purify and stabilize macromolecular complexes for single particle cryo-electron microscopy, to identify interactions between splicing factors that bind transiently to the spliceosome complex. This method is based on the centrifugation of samples into an increasing concentration of a fixation reagent to stabilize complexes. After centrifugation of yeast total extracts loaded on glycerol gradients, recovered fractions are analyzed by dot blot for the identification of the spliceosome sub-complexes and determination of the presence of individual splicing factors.

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Here, we describe the utilization of Grafix (Gradient Fixation), a glycerol gradient centrifugation in the presence of a crosslinker, to identify interactions between splicing factors that bind transiently to the spliceosome complex.

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Biology

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bis(sulfosuccinimidyl) Suberate (BS3)

Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for isolation of protein complexes under physiological conditions. Fusion proteins containing two copies of Strep-tag II, designated twin-Strep-tag or SIII-tag, have the advantage of higher affinity for Strep-Tactin compared to those containing only a single Strep-tag, thus allowing more efficient protein purification. However, this advantage is offset by the fact that elution of twin-Strep-tagged proteins with biotin may be incomplete, leading to low protein recovery. The recovery can be dramatically improved by using denaturing elution with sodium dodecyl sulfate (SDS), but this leads to sample contamination with Strep-Tactin released from the resin, making the assay incompatible with downstream proteomic analysis. To overcome this limitation, we have developed a method whereby resin-coupled tetramer of Strep-Tactin is first stabilized by covalent cross-linking with Bis(sulfosuccinimidyl) suberate (BS3) and the resulting cross-linked resin is then used to purify target protein complexes in a single batch purification step. Efficient elution with SDS ensures good protein recovery, while the absence of contaminating Strep-Tactin allows downstream protein analysis by mass spectrometry. As a proof of concept, we describe here a protocol for purification of SIII-tagged viral protein VPg-Pro from nuclei of virus-infected N. benthamiana plants using the Strep-Tactin polymethacrylate resin cross-linked with BS3. The same protocol can be used to purify any twin-Strep-tagged protein of interest and characterize its physiological binding partners.

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3

A method is described for efficient purification of twin-Strep-tagged fusion proteins and their specific complexes on modified streptavidin (Strep-Tactin) resin covalently cross-linked with Bis(sulfosuccinimidyl) suberate (BS3). The method has the advantages of fast speed, good target protein recovery and high purity, and is compatible with subsequent analysis by mass spectrometry.

Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for ...

Combining Chemical Cross-linking and Mass Spectrometry of Intact Protein Complexes to Study the Architecture of Multi-subunit Protein Assemblies

Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is therefore fundamental for the understanding of cellular processes. However, many protein complexes are dynamic assemblies and are not accessible by conventional structural techniques. Mass spectrometry contributes to the structural investigation of these assemblies, and particularly the combination of various mass spectrometric techniques delivers valuable insights into their structural arrangement.
In this article, we describe the application and combination of two complementary mass spectrometric techniques, namely chemical cross-linking coupled with mass spectrometry and native mass spectrometry. Chemical cross-linking involves the covalent linkage of amino acids in close proximity by using chemical reagents. After digestion with proteases, cross-linked di-peptides are identified by mass spectrometry and protein interactions sites are uncovered. Native mass spectrometry on the other hand is the analysis of intact protein assemblies in the gas phase of a mass spectrometer. It reveals protein stoichiometries as well as protein and ligand interactions. Both techniques therefore deliver complementary information on the structure of protein-ligand assemblies and their combination proved powerful in previous studies.

The architecture of protein complexes is essential for their function. Combining various mass spectrometric techniques proved powerful to study their assembly. We provide protocols for chemical cross-linking and native mass spectrometry and show how these complementary techniques help to elucidate the architecture of multi-subunit protein assemblies.

Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is ...

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

Source: Rotoli, S. M. et al., <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/59483">Combining Non-reducing SDS-PAGE Analysis and Chemical Crosslinking to Detect Multimeric Complexes Stabilized by Disulfide Linkages in Mammalian Cells in Culture</a>. 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.

Source: Rotoli, S. M. et al., Combining Non-reducing SDS-PAGE Analysis and Chemical Crosslinking to Detect Multimeric Complexes Stabilized by Disulfide ...

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

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