Supported by the NIH/NIDA DA034783. We thank Bryan A. Killinger for technical assistance with the multimer-PAGE.

1. Tissue Preparation
<ol>
	<li>Prepare 10 mL of 4x BN-PAGE sample buffer (200 mM Bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (Bis-Tris), 200 mM NaCl, 40% w/v glycerol, 0.004% Ponceau S, pH 7.2). 
	NOTE: This stock solution may be made in advance and stored at 4 &#176;C.</li>
	<li>Dilute 250 &#181;L of 4x sample buffer in 750 &#181;L dH2O containing 1x commercial protease inhibitor cocktail. Vortex and chill on ice.</li>
	<li>Homogenize 20 mg of target tissue in the 1 mL 1x ice-cold BN-PAGE sample buffer with 30 strokes of a clean dounce homogenizer. 
	NOTE: For this demonstration experiment, the target tissue is whole rat brain tissue. After homogenization, samples may be treated with mild detergent such as 2% digitonin to solubilize and permit electrophoresis of membrane proteins.</li>
	<li>Transfer the homogenate to a 1.5 mL microcentrifuge tube, and centrifuge at 14,000 x g for 30 min to pellet insoluble cellular contents. After centrifugation, decant the supernatant into a clean tube on ice.</li>
	<li>Follow manufacturer instructions to measure supernatant protein concentration using bicinchoninic acid (BCA) or similar protein quantitation assay.</li>
	<li>If the homogenate sample(s) contain detergent, add a quantity of 5% Coomassie Blue G-250 in aqueous solution sufficient to bring the homogenate solution to 0.25% Coomassie.</li>
</ol>
2. BN-PAGE
<ol>
	<li>Dilute 25 mL 20x blue native (BN) electrophoresis buffer stock (1.0 M Bis-Tris, 1.0 M tricine, pH 6.8) into 475 mL dH2O containing 0.002% Coomassie Blue to make 500 mL of 1x running buffer.
	<ol>
		<li>If samples contain detergent, instead make 250 mL of 1x running buffer containing 0.02% Coomassie and another 250 mL containing none.</li>
	</ol>
	</li>
	<li>Chill buffer(s) to 4 &#176;C.</li>
	<li>Clean and assemble a polyacrylamide gel pouring cassette according to manufacturer instructions.</li>
	<li>Pour BN polyacrylamide gel (3% T stacking layer, 6% T resolving layer) according to a standard recipe7, and place the well comb.</li>
	<li>After the gels have polymerized, rinse the cassette with dH2O and assemble the electrophoresis apparatus according to manufacturer instructions.</li>
	<li>Remove the well comb and fill the wells with 1x BN-PAGE running buffer. Avoid filling the entire inner chamber with buffer until samples are loaded.
	<ol>
		<li>If samples contain detergent, fill the wells with the buffer containing 0.02% Coomassie Blue. 
		NOTE: Perform steps 2.7-2.9 at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Using gel-loading tips, pipette a volume of homogenate containing 20 &#181;g of protein into the desired wells. Pipette an equivalent volume of 1x BN-PAGE sample buffer into any unused wells. 
	NOTE: Use the protein concentration determined from the BCA assay to calculate the appropriate volume of homogenate to add to the wells.</li>
	<li>After samples are loaded, fill the inner chamber with 1x BN-PAGE running buffer. Be sure the gel is entirely submerged in buffer. Next, fill the outer chamber with 1x running buffer to the level indicated by the manufacturer.
	<ol>
		<li>If samples contain detergent, fill the inner chamber with the 1x buffer containing 0.02% Coomassie Blue, and the outer chamber with dye-free 1x running buffer.</li>
	</ol>
	</li>
	<li>Connect the electrodes to the power supply, and electrophorese the proteins in the gel at 150 V until the dye band progresses ~2 cm into resolving layer. Stop and disconnect the power supply.</li>
</ol>
3. Cross-linking
NOTE: Perform Steps 3.1-3.6 at 4 &#176;C.
<ol>
	<li>Disassemble the electrophoresis apparatus, and separate the glass panes of the gel cassette. Remove and discard the stacking layer.</li>
	<li>Carefully cut the gel just below the bottom edge of the dye front. Take care to make this cut as smooth and straight as possible, and then discard the unused piece of gel. This will leave a ~2 cm wide, horizontal strip of polyacrylamide gel, which will contain all the protein in the homogenate sample.</li>
	<li>Trim away any unused portions along the edges of the gel strip.</li>
	<li>Carefully place the strip into 10 mL phosphate buffered saline (PBS) in a small container, and gently mix by nutation for 30 min to equilibrate.</li>
	<li>After equilibration, discard and replace the PBS with another 10 mL. Pipette 500 &#181;L of 25 mM DSP dissolved in dimethyl sulfoxide into the PBS, and continue mixing as above (step 3.4) for 30 min.</li>
	<li>Pour off the DSP solution. Add 10 mL of 0.375 M tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), pH 8.8, containing 2% sodium dodecyl sulfate (SDS) to quench the unreacted DSP. Continue nutation for 15 min.</li>
</ol>
4. SDS-PAGE
<ol>
	<li>While gel strip is quenching, prepare SDS-PAGE gel solutions according to standard methods9. Do not add polymerization reagents.</li>
	<li>After quenching, return the &#914;&#925; gel strip to room temperature, and cast the strip into a new gel cassette.
	<ol>
		<li>To do this, carefully pick up the gel and place it onto a clean gel cassette spacer plate.</li>
		<li>Orient the strip so the bottom of the dye front is nearest to the top of new cassette (i.e., the side that travelled furthest during BN-PAGE should be closest to the top of the new cassette; the gel strip should be flipped from its prior orientation). See Figure 3.</li>
		<li>Place the strip such that its top edge lies even with where the top edge of the cover plate will be (i.e., it will be at the top of the new gel). Make sure the dye front is parallel to the horizontal edges of the glass plate.</li>
		<li>Push one side of the excised strip against one of the spacer walls, leaving room on the other side for gel to be poured and a protein standard or ladder to be loaded.</li>
		<li>If the bottom edge of the gel strip contains any jagged or uneven areas, carefully cut them away. If present, they will trap bubbles during gel pouring.</li>
		<li>Once the gel strip is positioned correctly, lay the cover plate over the spacer plate. Apply gentle pressure to push out any trapped air bubbles.</li>
		<li>Continue to assemble the gel-pouring apparatus according to manufacturer instructions.</li>
	</ol>
	</li>
	<li>Add polymerization reagents to the resolving gel buffer, and pour it into the prepared gel cassette, using a serological pipette. Fill the gel cassette to ~2 cm below the excised BN-PAGE gel strip, to leave room for the stacking layer.</li>
	<li>Add 100 &#181;L butanol over the top of the poured gel, and allow 30 min for polymerization of the resolving layer. Pour off the butanol.</li>
	<li>Add polymerization reagents to the stacking gel solution. Using a serological pipette, pour the stacking layer to fill all remaining empty space in the gel cassette.
	<ol>
		<li>Tilt the gel cassette as the stacking layer is poured so it fills the space below the gel strip, and air bubbles are not trapped.</li>
		<li>As the stacking gel buffer fills the empty space below the gel strip, gradually return the gel cassette to level footing.</li>
		<li>Continue to fill the empty space next to the excised gel strip with the stacking gel buffer, until it nearly overflows.</li>
	</ol>
	</li>
	<li>Allow the stacking layer to polymerize for 30 min. 
	NOTE: Make 10x SDS-PAGE running buffer (250 mM tris(hydroxymethyl)aminomethane (tris), 1.9 M glycine, 1% SDS) while the gel is polymerizing. This can also be made beforehand and stored at room temperature.</li>
	<li>Dilute 50 mL 10X SDS-PAGE running buffer stock into 450 mL dH2O to make 1x working buffer.</li>
	<li>After the stacking layer has polymerized, remove the gel cassette from the pouring apparatus, rinse with dH2O, and assemble the electrophoresis apparatus according to manufacturer instructions.</li>
	<li>Fill the inner chamber completely with 1x SDS-PAGE running buffer, then fill the outer chamber to the level indicated by the manufacturer.</li>
	<li>Load the space next to the excised gel strip with a molecular weight ladder or appropriate protein standard.</li>
	<li>Attach the electrodes to the power supply, and electrophorese the samples at 120 V. When the Coomassie dye runs off the gel, stop and disconnect the power supply.</li>
	<li>Analyze the gel using standard methods of electroblotting and protein detection by antibody binding10.</li>
</ol>

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F., 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=Towards+a+proteome-scale+map+of+the+human+protein-protein+interaction+network.">Towards a proteome-scale map of the human protein-protein interaction network.</a> Nature. 437 (7062), 1173-1178 (2005).</li><li>Phizicky, E. M., Fields, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-protein+interactions:+methods+for+detection+and+analysis.">Protein-protein interactions: methods for detection and analysis.</a> Microbiol Rev. 59 (1), 94-123 (1995).</li><li>Ho, Y., 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=Systematic+identification+of+protein+complexes+in+Saccharomyces+cerevisiae+by+mass+spectrometry.">Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry.</a> Nature. 415 (6868), 180-183 (2002).</li><li>Krogan, N. 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=Global+landscape+of+protein+complexes+in+the+yeast+Saccharomyces+cerevisiae.">Global landscape of protein complexes in the yeast Saccharomyces cerevisiae.</a> Nature. 440 (7084), 637-643 (2006).</li><li>Wittig, I., Braun, H. P., Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+PAGE.">Blue native PAGE.</a> Nat Protoc. 1 (1), 418-428 (2006).</li><li>Schagger, H., Cramer, W. A., von Jagow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+molecular+masses+and+oligomeric+states+of+protein+complexes+by+blue+native+electrophoresis+and+isolation+of+membrane+protein+complexes+by+two-dimensional+native+electrophoresis.">Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis.</a> Anal Biochem. 217 (2), 220-230 (1994).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227 (5259), 680-685 (1970).</li><li>Beisiegel, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Blotting.">Protein Blotting.</a> Electrophoresis. 7 (1), 1-18 (1986).</li><li>Killinger, B. A., Moszczynska, A. <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+alpha-Synuclein+Multimer+Stoichiometry+in+Complex+Biological+Samples+by+Electrophoresis.">Characterization of alpha-Synuclein Multimer Stoichiometry in Complex Biological Samples by Electrophoresis.</a> Anal Chem. 88 (7), 4071-4084 (2016).</li><li>Newman, A. J., Selkoe, D., Dettmer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+quantitative+immunoblotting+of+endogenous+alpha-synuclein.">A new method for quantitative immunoblotting of endogenous alpha-synuclein.</a> PLoS One. 8 (11), e81314 (2013).</li><li>Wong, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemistry of protein conjugation and cross-linking. , (1991).</li><li>Seddon, A. M., Curnow, P., Booth, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+proteins,+lipids+and+detergents:+not+just+a+soap+opera.">Membrane proteins, lipids and detergents: not just a soap opera.</a> Biochim Biophys Acta. 1666 (1-2), 105-117 (2004).</li><li>Tanford, C., Reynolds, J. A. <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+membrane+proteins+in+detergent+solutions.">Characterization of membrane proteins in detergent solutions.</a> Biochim Biophys Acta. 457 (2), 133-170 (1976).</li><li>Peters, K., Richards, F. 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The authors have nothing to disclose.

Protein-protein interactions are important for every task living things carry out. Because of this, they are the subject of intense scrutiny and research. Multimer-PAGE is a novel method for capturing, separating, and analyzing a wide range of protein complexes. We have previously demonstrated its applicability to studying oligimerization of the disease-associated protein &#945;-synuclein11. However, it is extensible to many protein complexes, as demonstrated in Figure 2. When com...

Normal cellular function is dependent on protein-protein interactions1,2. As a result, human diseases are often marked by perturbations in the assembly and behavior of various protein complexes3. The ability to characterize such interactions is therefore critical. Current means of detecting these interactions require purification of target proteins, often followed by pull-down of their interacting partners. Conventional purification is accomplished by differential centrifugation, precipitation, and/or chromatography4. These methods are time-consuming, must be altered for each target protein, and often result in low yields. Modern purification methods involve the fusion of peptide tags to target proteins, followed by immunoprecipitation or extraction on columns loaded with beads bound to a capture molecule5,6. While this is extensible to many proteins, it requires sequence modification of the target, potentially resulting in constructs that differ in affinity for their usual binding partners. The delicate nature of some protein-protein interactions means this method may not be applicable to many scenarios. Additionally, the pull-down assays used to map protein-protein interactions may not capture an accurate cellular picture, due to restricted degrees of freedom and non-native levels of the bait protein.
Ideally, protein complexes could be detected in their native states, without the need for purification or pull-down. Blue native polyacrylamide gel electrophoresis (BN-PAGE) was developed as a less-denaturing alternative to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and allows for the separation of some proteins and complexes from biological samples7. However, proteins in BN-PAGE separate based on a large number of variables, including size, charge, three-dimensional structure, and association with other molecules. The interactions of these factors often result in co-separation of proteins, aggregate formation, and poor protein band resolution. Two-dimensional native-polyacrylamide gel electrophoresis resolves some, but not all, of these problems8.
To circumvent the complications associated with native separation, some authors use amine-reactive cross-linking reagents, such as dithiobis(succinimidyl propionate) (DSP), to capture protein complexes in tissue lysates4. These treated lysates can then be denatured and separated by SDS-PAGE, while preserving the native size and makeup of protein complexes. However, since cross-linking reagents react based on proximity of one molecule to another, and proteins in tissue lysates have many degrees of freedom and can stochastically interact, nonspecific background cross-linking can be high, especially in concentrated samples. This can lead to difficult-to-interpret results.
Here, we demonstrate the use of a hybrid BN-PAGE/SDS-PAGE method, termed multimer-polyacrylamide gel electrophoresis (multimer-PAGE), to separate and detect protein assemblies in complex mixtures. Initially, cell lysate is suspended in polyacrylamide gel via BN-PAGE. The lysate-containing gel is then reacted with the cross-linking reagent DSP. Pseudo-immobilized and slightly separated on the gel, proteins are much less likely to react nonspecifically, meaning background cross-linker reactivity is reduced. After cross-linking, the gel bands are excised and separated via SDS-PAGE. The resultant gel can then be analyzed by any means typically associated with polyacrylamide gel electrophoresis. This method allows for the separation and detection of native protein complexes in unmodified tissue lysate, without the need for additional purification or pull-down.

<table><tbody><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;epsilon;-Aminocaproic acid</td><td>Sigma</td><td>A2504</td><td></td></tr><tr><td>Acrylamide</td><td>Acros Organics</td><td>164855000</td><td>Toxic.</td></tr><tr><td>Acrylamide/bisacrilamide 37.5:1 (40%T stock solution)</td><td>BioRad</td><td>161-0148</td><td>Toxic.</td></tr><tr><td>Ammonium persulfate</td><td>Sigma</td><td>A3678</td><td></td></tr><tr><td>Anti rabbit IgG-HRP from goat</td><td>Santa Cruz Biotechnology</td><td>sc-2004</td><td></td></tr><tr><td>Bicinchoninic acid assay kit</td><td>Thermofisher</td><td>23225</td><td></td></tr><tr><td>Bis-tris</td><td>Sigma</td><td>B9754</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A9647</td><td></td></tr><tr><td>Butanol</td><td>Fisher Scientific</td><td>A399-1</td><td></td></tr><tr><td>Chemiluminescence substrate kit</td><td>ThermoFisher</td><td>24078</td><td></td></tr><tr><td>Coomassie blue G-250</td><td>Sigma-Aldrich</td><td>B0770</td><td></td></tr><tr><td>Digitonin</td><td>Sigma</td><td>D141</td><td>Toxic.</td></tr><tr><td>Dimethyl sulfoxide</td><td>Fisher Scientific</td><td>D128-1</td><td></td></tr><tr><td>Dithiobis(succinimidylpropionate)</td><td>Thermo Scientific</td><td>22585</td><td></td></tr><tr><td>Dry nonfat milk</td><td>LabScientific</td><td>M0841</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G9012</td><td></td></tr><tr><td>Glycine</td><td>Fisher Scientific</td><td>BP381-5</td><td></td></tr><tr><td>Halt Protease Inhibitor Cocktail</td><td>Thermofisher</td><td>78430</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Fisher Scientific</td><td>A144SI-212</td><td>For titration. Caustic.</td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A412-4</td><td>For PVDF membrane activation. Toxic.</td></tr><tr><td>Monoclonal anti &amp;alpha;-synuclein IgG from rabbit</td><td>Santa Cruz Biotechnology</td><td>sc-7011-R</td><td></td></tr><tr><td>N,N,N&#039;,N&#039;-tetramethylethylenediamine</td><td>Sigma</td><td>T9281</td><td></td></tr><tr><td>N,N&#039;-methylenebisacrylamide</td><td>Acros Organics</td><td>16479</td><td>Toxic.</td></tr><tr><td>NP40</td><td>Boston Bioproducts</td><td>P-872</td><td></td></tr><tr><td>Polysorbate 20 (tween-20)</td><td>Fisher Scientific</td><td>BP337-500</td><td></td></tr><tr><td>Polyvinylidene fluoride transfer membranes</td><td>Thermo Scientific</td><td>88518</td><td></td></tr><tr><td>Ponceau S</td><td>Sigma</td><td>P3504</td><td></td></tr><tr><td>Potassium chloride</td><td>Fisher Scientific</td><td>P217-3</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma</td><td>P9791</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Thermo Scientific</td><td>88265</td><td></td></tr><tr><td>SDS solution (10% w/v)</td><td>BioRad</td><td>161-0416</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher Scientific</td><td>BP358-212</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma</td><td>L37771</td><td></td></tr><tr><td>Sodium phosphate monobasic</td><td>Fisher Scientific</td><td>BP329-1</td><td></td></tr><tr><td>Tricine</td><td>Sigma</td><td>T0377</td><td></td></tr><tr><td>Tris base</td><td>Fisher Scientific</td><td>BP152-500</td><td></td></tr><tr><td>Tris-HCl (0.5 M buffer, pH 6.8)</td><td>BioRad</td><td>161-0799</td><td></td></tr><tr><td>Tris-HCl (1.5 M buffer, pH 8.8)</td><td>BioRad</td><td>161-0798</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GE Imagequant LAS 4000</td><td>GE Healthcare</td><td>28-9558-10</td><td></td></tr><tr><td>ImageJ software</td><td>NIH</td><td></td><td></td></tr><tr><td>Synergy H1 microplate reader</td><td>BioTek</td><td></td><td></td></tr><tr><td>Gel Former + Stand</td><td>Biorad</td><td></td><td></td></tr><tr><td>Microfuge 22R centrifuge</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>2 mL dounce homogenizer</td><td></td><td></td><td></td></tr><tr><td>Vortex mixer</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Ultrasonic tissue homogenizer</td><td>Fisher Scientific</td><td>FB120220</td><td></td></tr></tbody></table>

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.

In this demonstration experiment, multimer-PAGE was performed on whole rat brain lysate. The resultant separated proteins were blotted onto polyvinylidene diflouride (PVDF) membranes, and then probed with antibodies against proteins that are known to form complexes. Figure 1 shows a validation of the protocol by two means. First, we demonstrate that the cross-linked proteins are cleavable by addition of a reducing agent, meaning the observed higher molecular weight specie...

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

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 ...

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11.3K Views.  Michigan State University. 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.

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

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed complexes. The latter can be elegantly resolved according to molecular size using native polyacrylamide gel electrophoresis (BN-PAGE). Coupling of such separations to sensitive mass spectrometry (BN-MS) has been well-established and theoretically allows for exhaustive assessment of the extractable complexome in biological samples. However, this approach is rather laborious and provides limited complex size resolution and sensitivity. Also, its application has remained restricted to abundant mitochondrial and plastid proteins. Thus, for a majority of proteins, information regarding integration into stable protein complexes is still lacking. Presented here is an optimized approach for complexome profiling comprising preparative-scale BN-PAGE separation, sub-millimeter sampling of broad gel lanes by cryomicrotome slicing, and mass spectrometric analysis with label-free protein quantification. The procedures and tools for critical steps are described in detail. As an application, the report describes complexome analysis of a solubilized endosome-enriched membrane fraction from mouse kidneys, with 2,545 proteins profiled in total. The results demonstrate identification of uniform, low-abundance membrane proteins such as intracellular ion channels as well as high resolution, complex protein assembly patterns, including glycosylation isoforms. The results are in agreement with independent biochemical analyses. In summary, this methodology allows for comprehensive and unbiased identification of protein (super)complexes and their subunit composition, providing a basis for investigating stoichiometry, assembly, and interaction dynamics of protein complexes in any biological system.

A versatile cryoslicing BN-MS protocol using a microtome is presented for high-resolution complexome profiling.

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed ...

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

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Immunology and Infection

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 ...

Multimer-PAGE for Separating Native Protein Complexes: A Hybrid Separation Technique Consisting of Blue Native-PAGE and SDS-PAGE to Separate Intact Multimeric Proteins From Tissue Lysate

Source: Rhinesmith, T. et al., <a href="https://www.jove.com/v/55341">Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples</a>. J. Vis. Exp. (2017).
This video describes multimeric PAGE for separating native protein complexes from tissue homogenates. The technique is a hybrid of blue native-PAGE and SDS-PAGE techniques. The separated complexes can be characterized and studied for their role in cell functioning.

Multimer-PAGE do separacji natywnych kompleksów białkowych: hybrydowa technika separacji składająca się z Blue native-PAGE i SDS-PAGE w celu oddzielenia nienaruszonych białek multimerycznych od lizatu tkankowego

Source: Rhinesmith, T. et al., Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples. J. Vis. Exp. (2017).
This ...

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

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Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Sheathless Capillary Electrophoresis–Mass Spectrometry for Metabolic Profiling of Biological Samples

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

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

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

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

Multimer-PAGE do separacji natywnych kompleksów białkowych: hybrydowa technika separacji składająca się z Blue native-PAGE i SDS-PAGE w celu oddzielenia nienaruszonych białek multimerycznych od lizatu tkankowego