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 samp...

<ol>
	<li>Alberts, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+as+a+collection+of+protein+machines:+preparing+the+next+generation+of+molecular+biologists.">The cell as a collection of protein machines: preparing the next generation of molecular biologists.</a> Cell. 92 (3), 291-294 (1998).</li><li>Pawson, T., Nash, P. <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+define+specificity+in+signal+transduction.">Protein-protein interactions define specificity in signal transduction.</a> Genes Dev. 14 (9), 1027-1047 (2000).</li><li>Rual, J. 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. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+cross-linking:+reagents+and+problems+in+studies+of+membrane+structure.">Chemical cross-linking: reagents and problems in studies of membrane structure.</a> Annu Rev Biochem. 46, 523-551 (1977).</li><li>Lomant, A. J., Fairbanks, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+probes+of+extended+biological+structures:+synthesis+and+properties+of+the+cleavable+protein+cross-linking+reagent+[35S]dithiobis(succinimidyl+propionate).">Chemical probes of extended biological structures: synthesis and properties of the cleavable protein cross-linking reagent [35S]dithiobis(succinimidyl propionate).</a> J Mol Biol. 104 (1), 243-261 (1976).</li><li>Sun, 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=Crystal+structure+of+mitochondrial+respiratory+membrane+protein+complex+II.">Crystal structure of mitochondrial respiratory membrane protein complex II.</a> Cell. 121 (7), 1043-1057 (2005).</li><li>Wittig, I., 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=Native+electrophoretic+techniques+to+identify+protein-protein+interactions.">Native electrophoretic techniques to identify protein-protein interactions.</a> Proteomics. 9 (23), 5214-5223 (2009).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="817">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Chemicals</td>
			<td style="width:195px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">&#949;-Aminocaproic acid</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">A2504</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Acrylamide</td>
			<td style="width:195px;">Acros Organics</td>
			<td style="width:119px;">164855000</td>
			<td>Toxic.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:336px;">Acrylamide/bisacrilamide 37.5:1 (40%T stock solution)</td>
			<td style="width:195px;">BioRad</td>
			<td style="width:119px;">161-0148</td>
			<td>Toxic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Ammonium persulfate</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">A3678</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Anti rabbit IgG-HRP from goat</td>
			<td style="width:195px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-2004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Bicinchoninic acid assay kit</td>
			<td style="width:195px;">Thermofisher</td>
			<td style="width:119px;">23225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Bis-tris</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">B9754</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Bovine serum albumin</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">A9647</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Butanol</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">A399-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Chemiluminescence substrate kit</td>
			<td style="width:195px;">ThermoFisher</td>
			<td style="width:119px;">24078</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Coomassie blue G-250</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:119px;">B0770</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Digitonin</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">D141</td>
			<td>Toxic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Dimethyl sulfoxide</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">D128-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Dithiobis(succinimidylpropionate)</td>
			<td style="width:195px;">Thermo Scientific</td>
			<td style="width:119px;">22585</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Dry nonfat milk</td>
			<td style="width:195px;">LabScientific</td>
			<td style="width:119px;">M0841</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Glycerol</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">G9012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Glycine</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">BP381-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Halt Protease Inhibitor Cocktail</td>
			<td style="width:195px;">Thermofisher</td>
			<td>78430</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Hydrochloric acid</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">A144SI-212</td>
			<td>For titration. Caustic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Methanol</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">A412-4</td>
			<td>For PVDF membrane activation. Toxic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Monoclonal anti &#945;-synuclein IgG from rabbit</td>
			<td style="width:195px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-7011-R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">N,N,N&#39;,N&#39;-tetramethylethylenediamine</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">T9281</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">N,N&#39;-methylenebisacrylamide</td>
			<td style="width:195px;">Acros Organics</td>
			<td style="width:119px;">16479</td>
			<td>Toxic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">NP40</td>
			<td style="width:195px;">Boston Bioproducts</td>
			<td style="width:119px;">P-872</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Polysorbate 20 (tween-20)</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">BP337-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Polyvinylidene fluoride transfer membranes</td>
			<td style="width:195px;">Thermo Scientific</td>
			<td style="width:119px;">88518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Ponceau S</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">P3504</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Potassium chloride</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">P217-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Potassium phosphate monobasic</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">P9791</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Protease inhibitor cocktail</td>
			<td style="width:195px;">Thermo Scientific</td>
			<td style="width:119px;">88265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">SDS solution (10% w/v)</td>
			<td style="width:195px;">BioRad</td>
			<td style="width:119px;">161-0416</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Sodium chloride</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">BP358-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Sodium dodecyl sulfate</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">L37771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Sodium phosphate monobasic</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">BP329-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Tricine</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">T0377</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Tris base</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">BP152-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Tris-HCl (0.5 M buffer, pH 6.8)</td>
			<td style="width:195px;">BioRad</td>
			<td style="width:119px;">161-0799</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Tris-HCl (1.5 M buffer, pH 8.8)</td>
			<td style="width:195px;">BioRad</td>
			<td style="width:119px;">161-0798</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Name</td>
			<td style="width:195px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Instruments</td>
			<td style="width:195px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">GE Imagequant LAS 4000</td>
			<td style="width:195px;">GE Healthcare</td>
			<td style="width:119px;">28-9558-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">ImageJ software</td>
			<td style="width:195px;">NIH</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Synergy H1 microplate reader</td>
			<td style="width:195px;">BioTek</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Gel Former + Stand</td>
			<td style="width:195px;">Biorad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Microfuge 22R centrifuge</td>
			<td style="width:195px;">Beckman Coulter</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">2 mL dounce homogenizer</td>
			<td style="width:195px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Vortex mixer</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Ultrasonic tissue homogenizer</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">FB120220</td>
			<td></td>
		</tr>
	</tbody>
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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 ...

The overall goal of this protocol is to enable researchers to capture, separate, and analyze native protein complexes extracted from tissue lysates using polyacrylamide gel electrophoresis. This method can aid researchers who study proteins because it is able to stabilize otherwise labile protein associations under near-native conditions, allowing for their identification and subsequent analysis. The main advantage of this technique is that it uses common biological methods so it is widely applicable and can be tailored to the goals of the individual researcher.To begin this procedure, prepare for blue native polyacrylamide gel electrophoresis as described in the text protocol. Connect the electrodes to the power supply and electrophorese the proteins in the gel at 150 volts until the dye band progresses approximately two centimeters into the resolving layer. At this point, stop and disconnect the power supply.After disassembling the electrophoresis apparatus, separate the glass panes of the gel cassette. Remove and discard the stacking layer. Carefully cut the gel just below the bottom edge of the dye front, taking care to make this cut as smooth and straight as possible.Then, discard the unused piece of gel. Trim away any unused portions along the edges of the gel strip. Carefully place the strip into 10 milliliters of phosophate buffered saline in a small container and gently mix by notation for 30 minutes to equilibrate.After equilibration, discard and replace the PBS with another 10 milliliters. Pipette 500 microliters of 25 millimolar DSP dissolved in dimethyl sulfoxide into the PBS and continue mixing as before for 30 minutes. After 30 minutes, pour off the DSP solution.At 10 milliliters of 0.375 molar HSCL, pH 8.8, containing 2%sodium dodecyl sulfate to quench the unreacted DSP. Continue the notation for 15 minutes. While the gel strip is quenching, prepare SDS-PAGE gel solutions according to standard methods.Do not add the polymerization reagents. After quenching, return the blue native gel strip to room temperature and cast the strip into a new gel cassette. To do this, carefully pick up the gel and place it onto a clean gel cassette spacer plate.Orient the strip so that it is flipped from its prior orientation and the bottom of the dye front is nearest to the top of the new cassette. Place the strip such that its top edge lies even with where the top edge of the cover plate will be. Make sure the dye front is parallel to the horizontal edges of the glass plate.Push one side of the excised strip against one of the spacer walls, leaving room on the other side for the gel to be poured and a protein standard or ladder to be loaded. If the bottom edge of the gel strip contains any jagged or uneven areas, carefully cut them away. 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. Continue to assemble the gel-pouring apparatus according to the manufacturer's instructions. The gel strip sometimes shifts when the top plate is being placed.It helps to let the strip hang over the edge of the top plate a little so it can be adjusted with a spatula to correct any shifts. 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 approximately two centimeters below the excised blue native PAGE gel strip to leave room for the stacking layer.Then, add 100 microliters of butanol over the top of the poured gel and allow 30 minutes for polymerization of the resolving layer before pouring off the butanol. Next, add the 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.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. As the stacking gel buffer fills the empty space below the gel strip, gradually return the gel cassette to level footing. Continue to fill the empty space next to the excised gel strip with the stacking gel buffer until it nearly overflows.Then, allow the stacking layer to polymerize for 30 minutes. After the stacking layer has polymerized, remove the gel cassette from the pouring apparatus, rinse it with distilled water, and assemble the electrophoresis apparatus according to the manufacturer's instructions. Fill the inner chamber completely with 1x SDS-PAGE running buffer.Then, fill the outer chamber to the level indicated by the manufacturer. Load the space next to the excised gel strip with a molecular weight ladder or the appropriate protein standard. Attach the electrodes to the power supply and electrophorese the samples at 120 volts.When the Coomassie dye runs off the gel, stop the run and disconnect the power supply. Analyze the gel using standard methods of electroblotting and antibody-based protein detection. Validation of multimer-PAGE is shown here via immunoblot.The captured kinesin complex is cleaved by diphyo-3-itol showing that the high molecular weight aggregates are formed by cross-linking smaller substituents. Depicted here, brain lysates treated with increasing concentrations of SDS or Triton increased detection of monomeric alpha-synuclein while decreasing the detection of the soluble oligomer tetromer. Here, representative results of multimer-PAGE used to capture soluble complexes in rat brain lysate are shown.Proteins were detected via immunoblot. The expected monomeric weight of each protein is shown below the lanes. Similarly, capture of membrane-bound complexes is shown here.Multimer-PAGE occasionally fails to capture a complex, as is shown on the SDHA respiratory complex two blot. A discussion of possible explanations for this can be found in the text protocol. Once mastered, this technique can be done in about eight hours if it is performed properly.After watching this video, you should have a good understanding of how to perform in-gel cross-linking of native protein complexes followed by gel re-casting and separation by secondary SDS-PAGE. Don't forget that working with acrylamide can be extremely hazardous and personal protective equipment should be worn while casting the gel. Following this procedure, the separated stabilized complexes can be eluted or cut from the gel and can be analyzed by many means, including immunodetection or mass spectrometry depending on the researcher's needs.

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Biochemistry

11.2K Görüntüleme.  Michigan State University. The overall goal of this protocol is to enable researchers to capture, separate, and analyze native protein complexes extracted from tissue lysates using polyacrylamide gel electrophoresis. This method can aid researchers who study proteins because it is able to stabilize otherwise labile protein associations under near-native conditions, allowing for their identification and subsequent analysis. The main advantage of this technique is that it uses common biological methods so it is widely ap...