This work was funded by a Grant from the Swiss National Science foundation (Grant nr. 31003A_143834) to JP.

NOTE: This protocol is based on the previously published non-denaturing PAGE protocol 12. In that study, the oligomeric state of the MxA protein was assessed using either Vero cells overexpressing MxA or IFN-&#945;-stimulated A549 cells expressing endogenous MxA. The protocol described below can be used to analyze the oligomeric state of any protein in addition to MxA. However, further optimization may be required.
1. Preparation of Cell Lysate for Non-denaturing PAGE
NOTE: To analyze the oligomeric state of the human MxA protein in either Vero or A549 cells, 1.0 x 106 cells were harvested. Depending on the cell type or the abundance of the protein to analyze, the cell number should be adjusted. It is also important to protect the lysis buffer from light exposure, as soon as the light-sensitive iodoacetamide is added.
<ol>
	<li>Seed 0.3 x 106 A549 or Vero cells per well into 6 well-dishes. Keep the cells in 2 ml growth medium per well (see Table 1). Incubate cells overnight in a cell culture incubator (37 &#176;C, 5% CO2).</li>
	<li>Harvest the cells by washing with 1 ml of phosphate buffered saline (PBS) and detach by adding 0.5 ml of 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) 1x solution for approximately 5 min at room temperature.</li>
	<li>As soon as the cells detach from the dish, add 0.5 ml growth medium and carefully mix by pipetting up and down.</li>
	<li>Transfer the cells of each well into one 2 ml tube and pellet them using a table top centrifuge (5,000 x g, 4 &#176;C, 5 min).</li>
	<li>Carefully remove the supernatant by pipetting without disturbing the cell pellet.</li>
	<li>Wash the cells with 1 ml ice-cold PBS by carefully pipetting the cell suspension up and down.</li>
	<li>Pellet cells in a table top centrifuge (5,000 x g, 4 &#176;C, 5 min).</li>
	<li>Carefully remove the supernatant by pipetting without detaching the cell pellet.</li>
	<li>Resuspend cells in 200 &#956;l ice-cold lysis buffer (see Table 1) by pipetting up and down and put on ice.</li>
	<li>Immediately, protect lysate from light by covering the tubes using tin foil and incubate for 30 min on ice. 
	NOTE: After incubation for 30 min on ice, it is no longer essential to protect the lysate from light exposure, since the protection of the free thiol groups is irreversible.</li>
	<li>Remove cell debris by centrifugation in a pre-chilled table top centrifuge (13,000 x g, 4 &#176;C, 20 min).</li>
	<li>Equilibrate dialysis columns in dialysis buffer (Table 1) in the cold room at 4 &#176;C for 20 min during the centrifugation step. Use a column with a molecular weight cut off of 10,000.
	<ol>
		<li>Attach the columns to a float buoy and put them into a beaker filled with dialysis buffer. To ensure gentle stirring, use a magnetic stirrer. Do not touch the membrane. 
		NOTE: Dialysis columns can be purchased or prepared from 1.5 ml tubes according to the protocol described by Fiala and coworkers 19.</li>
	</ol>
	</li>
	<li>Remove the columns from the dialysis buffer and the float buoy. Transfer the cleared lysates into the prepared dialysis column by pipetting without touching the membrane. Attach the columns to a float buoy and put them back into the beaker filled with dialysis buffer.</li>
	<li>Dialyze the lysate in a beaker containing ice-cold dialysis buffer (Table 1) for at least 4 hr (or preferably overnight) at 4 &#176;C while carefully stirring using a magnetic stirrer. Use at least 100 ml dialysis buffer for a 200 &#181;l lysate.</li>
	<li>Transfer the dialysed sample into a 1.5 ml tube. Remove precipitates by centrifugation in a table top centrifuge (13,000 x g, 4 &#176;C, 20 min). To prevent the dissociation of the oligomeric protein complexes continue with the protocol (section 2) immediately after dialysis. Do not freeze the prepared lysates.</li>
</ol>
2. Electrophoresis
NOTE: Electrophoresis was performed as described before with some modifications 22. In the protocol described below, pre-cast gradient gels were used (4-15% gradient). Alternatively, the gels can be prepared in the laboratory. It is very important to exclude any denaturing agent such as SDS to prevent the dissociation of the oligomeric protein complexes. Time of electrophoresis was optimized for the different oligomeric states of the human MxA protein. However, it can vary for other proteins, depending on the size of the oligomeric complex as well as the range of separation that is supposed to be achieved to analyze the complex. Therefore, the optimal time of electrophoresis should be determined empirically. For optimal resolution of the oligomers to be analyzed the current should not exceed 25 mA.
<ol>
	<li>Assemble the non-denaturing PAGE gel in the gel chamber. Fill the inner and outer chamber with pre-chilled running buffer (Table 1).</li>
	<li>Pre-run the gel with pre-chilled running buffer at 25 mA per gel for 15 min in the cold room at 4 &#176;C.</li>
	<li>Mix 15 &#181;l of the above prepared lysates with 5 &#181;l of 4x sample buffer (Table 1). Do not boil the sample.</li>
	<li>Load 15 &#181;l of sample and a native protein standard of choice on the gel. Run the gel at 25 mA for 4 hr in the cold room at 4 &#176;C. 
	NOTE: For semi-quantitative analyses, a protein quantification protocol (e.g. a Bradford protein assay 23) can be performed in order to ensure loading of equal amounts of total protein per lane.</li>
</ol>
3. Western Blot
NOTE: Described below is the protocol of a wet western blot system. Any blotting membrane can be used. Activate polyvinylidene fluoride (PVDF) membranes in 100% methanol before equilibration in blotting buffer. The Semi-dry western blot technique can be used alternatively, but has to be optimized for large oligomeric complexes.
<ol>
	<li>Disassemble the gel and carefully transfer it into SDS buffer (Table 1).</li>
	<li>Incubate for 10 min at room temperature while gently shaking.</li>
	<li>Prepare 2 sponges, 4 cellulose filter paper sheets and a blotting membrane per gel. Soak them in blotting buffer (Table 1).</li>
	<li>Assemble the sandwich as follows (bottom to top): 1 sponge, 2 cellulose filter paper sheets, membrane, gel, 2 cellulose filter paper sheets, 1 sponge.</li>
	<li>Put the sandwich into the blotting tank. Make sure that the membrane faces the plus pole while the gel faces the minus pole.</li>
	<li>Fill the blotting tank with pre-chilled blotting buffer.</li>
	<li>Blot at 90 mA overnight at 4 &#176;C for best protein transfer results.</li>
	<li>Disassemble the sandwich and visualize the protein standard by incubating the membrane in Ponceau S solution for 5 min at room temperature.</li>
	<li>Destain the membrane by carefully washing off the Ponceau S with deionized water until you can clearly see the bands of the protein standard.</li>
	<li>Mark the bands of the protein standard using a pen. 
	NOTE: Residual Ponceau S can interfere with the immunostaining. To avoid this, the membrane can be destained further by incubation in 0.1 M NaOH for 1 min and subsequent washing with deionized water.</li>
	<li>Block the membrane with Blocking buffer (see Table 1) for at least 1 hr at room temperature or overnight at 4 &#176;C.</li>
	<li>Visualize protein(s) of interest by immunostaining using antibodies directed against the protein to be analyzed. 
	NOTE: The human MxA protein was visualized using the rabbit polyclonal antibody specific for human Mx1 diluted 1:1,000 in Blocking buffer (Table 1). The antibody solution was incubated overnight at 4 &#176;C. Alternatively, the monoclonal anti-MxA antibody (clone 143) can be used (data not shown) 24.</li>
</ol>

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</ol>

The authors have nothing to disclose.

Here we describe a simple method that allows the rapid determination of the oligomeric state of proteins expressed in mammalian cells by non-denaturing PAGE followed by Western blot analysis. The major advantage of this approach is that the oligomeric state of a given protein can be determined from whole cell lysates without prior protein purification. This may be important for proteins that oligomerize or exert their function in association with auxiliary factors. In addition, the proteins are still in their native stat...

The quaternary structure of a protein plays a crucial role in many cellular processes. Signaling pathways, gene expression, and enzyme activation/deactivation all rely on the proper assembly of protein complexes 1-4. This process also known as homo- or hetero-oligomerization is due to irreversible covalent or reversible electrostatic and hydrophobic protein-protein interactions. Oligomerization not only diversifies the different cellular processes without increasing the genome size, but also provides a strategy for proteins to build stable complexes that are more resistant towards denaturation and degradation 5. Defects in oligomerization have an impact on the function of proteins and can lead to the development of diseases. For example, the enzyme phenylalanine hydroxylase forms a tetrameric complex. Some mutations within the protein complex can weaken the tetramer formation and lead to the disease phenylketonuria 6.
The human MxA protein is an interferon (IFN)-induced antiviral effector protein exerting a broad antiviral activity against various RNA as well as DNA viruses 7. It belongs to the superfamily of dynamin-like large GTPases and has the capacity to form large oligomeric structures in vitro 8. Oligomerization has been suggested to protect MxA from rapid degradation 9,10. Despite intense efforts by many research groups, the molecular mechanism of action remains largely elusive and the role of the oligomerization state of MxA for its antiviral function is under debate 9,11,12. In this regard, Gao and coworkers proposed a model where MxA exerts its antiviral activity by interacting with viral nucleoproteins in form of large ring-like oligomeric structures 11. However, more recently, we demonstrated that MxA dimers exhibit antiviral activity and interact with the nucleoprotein of influenza A virus 12. Based on the crystal structure of MxA, Gao and coworkers identified several amino acid residues in the interface regions that are critical for its oligomerization in vitro and its antiviral function 11,13. Therefore, in order to elucidate which oligomeric state of MxA exerts antiviral activity, we sought to establish a simple protocol to rapidly determine the oligmeric state of MxA interface mutants expressed in human cells as well as endogenous MxA expressed after IFN&#945; stimulation.
Although there are many techniques that are commonly used to investigate the interaction between proteins such as the split-Green Fluorescent Protein (split-GFP) complementation assay 14, surface plasmon resonance 15 and F&#246;rster resonance energy transfer (FRET) 16, they do not provide information of the exact stoichiometry of an oligomeric protein complex. For investigation of this particular aspect, techniques such as multi-angle light scattering (MALS) 17 and analytical ultracentrifugation 18 are very useful. Usually, the proteins analyzed using these methods are purified proteins. Oligomerization processes may also depend on other cellular factors. If these factors are unknown, the analysis is more difficult. Additionally, some proteins are difficult to express in E. coli and to purify. Therefore, these methods are not the optimal choice to analyze protein oligomerization in the cellular environment. In addition, these techniques require expensive instruments which are not readily available.
Non-denaturing polyacrylamide gel electrophoresis (PAGE), size exclusion chromatography or chemical crosslinking followed by conventional Sodium dodecyl sulfate (SDS)-PAGE are useful tools for the characterization of the formation of oligomers from cell lysates 2,19,20. These methods do not require specialized equipment and can be easily performed in a standard laboratory. We initially evaluated various chemical cross-linking protocols that invariantly led to non-specific aggregation and precipitation of MxA. Therefore, we next tested non-denaturing PAGE protocols. As non-denaturing PAGE excludes the use of SDS, the migration of proteins depends on their native charge. Blue-native PAGE uses coomassie brilliant blue G250 to load proteins with an overall negative charge, similar to SDS, but does not denature the protein 21. Unfortunately, coomassie brilliant blue precipitates in the presence of high salts and divalent cations (e.g. Mg2+) that are often included in lysis buffers. Depending on the buffers used, it may be difficult to analyze the sample without further optimization of steps that could have an effect on the oligomeric protein complex.
Here we present a simple protocol based on a previously published method 22 to determine oligomerization of human MxA protein derived from cellular lysates using non-denaturing PAGE.

<table><tbody><tr><td>Slide-A-Lyzer MINI Dialysis Units, 10K MWCO, 0.5 ml</td><td>Thermo Fisher Scientific</td><td>69570</td><td>Pre-equilibrate in dialysis buffer (if Glycerol removal is desired)&lt;br/&gt; Can be self-made according to Fiala et al. 2011</td></tr><tr><td>4&amp;ndash;15% Mini-PROTEAN TGX Precast Protein Gels, 10-well,</td><td>Bio-Rad</td><td>456-1083</td><td>Pre-run in running buffer to adjust buffer system</td></tr><tr><td>cOmplete, Mini, EDTA-free</td><td>Roche&amp;nbsp;</td><td>11836170001</td><td>use 1 tablet per 50 ml</td></tr><tr><td>PBS, pH 7.4&amp;nbsp; bottle a 500 ml Gibco</td><td>Thermo Fisher Scientific</td><td>14190-094</td><td></td></tr><tr><td>Ponceau S solution</td><td>Sigma-Aldrich</td><td>P7170</td><td>&lt;strong&gt;TOXIC&lt;/strong&gt; wear gloves and protect eyes</td></tr><tr><td>NativeMark Unstained Protein Standard&amp;nbsp; 50 &amp;micro;l</td><td>Invitrogen</td><td>P/N 57030</td><td>load 5 &amp;micro;l/well</td></tr><tr><td>A549 cells</td><td>ATCC</td><td>ATCC CCL185</td><td>Grow in growth medium (see &lt;strong&gt;Table 1&lt;/strong&gt;)</td></tr><tr><td>Vero cells</td><td>ATCC</td><td>ATCC CCL81</td><td>Grow in growth medium (see &lt;strong&gt;Table 1&lt;/strong&gt;)</td></tr><tr><td>anti-Mx1 antibody</td><td>Novus Biologicals</td><td>H00004599_D01P</td><td>Use at a 1:1,000 dilution</td></tr><tr><td>ECL Anti-rabbit IgG, Horseradish Peroxidase linked whole antibody (from donkey)</td><td>GE-Healthcare</td><td>NA934V</td><td>Use at a 1:10,000 dilution</td></tr><tr><td>0.5% Trypsin-EDTA (1x)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Life Technologies</td><td>Thermo Fisher</td><td>15400-054</td><td></td></tr><tr><td>Iodoacetamide&amp;nbsp;&amp;nbsp; 5 g</td><td>Sigma-Aldrich</td><td>I-6125</td><td>stock&amp;nbsp; 100 mM</td></tr><tr><td>Bromphenolblue</td><td>Sigma-Aldrich</td><td>B0126-25G</td><td></td></tr><tr><td>DMEM +4.5g/l Gluc,+L-Glut,+Pyruvate life technologies</td><td>Thermo Fisher Scientific</td><td>41966-029</td><td></td></tr><tr><td>Pen&amp;nbsp; Strep 100 x&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 100ml&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; life technologies</td><td>Thermo Fisher Scientific</td><td>15140 - 130</td><td></td></tr><tr><td>Glutamax 100x Stock, 100 ml&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; life technologies</td><td>Thermo Fisher Scientific</td><td>350500-038</td><td></td></tr><tr><td>Fetal Bovine Serum, Dialyzed , US Origin 500 ml Gibco Lot:42G9552K</td><td>Thermo Fisher Scientific</td><td>10270-106</td><td></td></tr><tr><td>Cellulose filter paper</td><td>Bio-Rad</td><td>1703965</td><td></td></tr><tr><td>PVDF blotting&amp;nbsp; membrane</td><td>GE-Healthcare</td><td>10600022</td><td></td></tr><tr><td>Tris(hydroxymethyl)aminomethane</td><td>Biosolve</td><td>0020092391BS</td><td></td></tr><tr><td>sodium fluoride (NaF)</td><td>Sigma Aldrich</td><td>S-7920</td><td></td></tr><tr><td>NP-40</td><td>Calbiochem</td><td>492015</td><td></td></tr><tr><td>cOmplete, Mini, EDTA-free</td><td>Roche&amp;nbsp;</td><td>11836170001</td><td></td></tr><tr><td>Tween 20</td><td>Calbiochem</td><td>6555204</td><td></td></tr><tr><td>CHAPS 10% solution</td><td>Amresco</td><td>N907</td><td></td></tr><tr><td>DL-Dithiothreitol (DTT)</td><td>Sigma Aldrich</td><td>43819</td><td></td></tr><tr><td>Glycine</td><td>Biosolve</td><td>0007132391BS</td><td></td></tr><tr><td>sodium orthovanadate (Na&lt;sub&gt;3&lt;/sub&gt;VO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>450243</td><td></td></tr><tr><td>Glycerol</td><td>Sigma Aldrich</td><td>G7757</td><td></td></tr><tr><td>&amp;#946;-Glycerophospate</td><td>Sigma Aldrich</td><td>G9422</td><td></td></tr><tr><td>Milk powder</td><td>Migros/Switzerland</td><td></td><td></td></tr><tr><td>Methanol</td><td>Millipore</td><td>1.06009</td><td></td></tr><tr><td>sodium cloride (NaCl)</td><td>Sigma Aldrich</td><td>71380</td><td></td></tr><tr><td>magnesium chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Amresco</td><td>288</td><td></td></tr><tr><td>Sodium dodecyl sulphate (SDS)</td><td>Sigma Aldrich</td><td>L4509</td><td></td></tr><tr><td>sodium hydroxide (NaOH)</td><td>Sigma Aldrich</td><td>S-8045</td><td></td></tr></tbody></table>

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.

Using non-denaturing PAGE, we analyzed the oligomeric state of the human wild type MxA, the dimeric interface mutants MxA(R640A) and MxA(L617D) as well as the monomeric interface mutant MxA(M527D) from cell lysates 12. Cells were lysed in a buffer containing 1% octylphenoxypolyethoxyethanol (NP-40) and iodoacetamide to ensure protein solubilization and protection of free thiol groups (see Figure 1). As described before, salt and small metabolites were removed by dialysis 19. Protein...

Watch this Scientific Journal Video about Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis at JoVE.com

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

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 ...

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Biochemistry

10.2K Views.  University of Zurich. 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.

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

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 ...

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 ...

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the cell, ATP. The ETC is composed of 5 massive protein complexes, which also assemble into supercomplexes called respirasomes (C-I, C-III, and C-IV) and synthasomes (C-V) that increase the efficiency of electron transport and ATP production. Various methods have been used for over 50 years to measure ETC function, but these protocols do not provide information on the assembly of individual complexes and supercomplexes. This protocol describes the technique of native gel polyacrylamide gel electrophoresis (PAGE), a method that was modified more than 20 years ago to study ETC complex structure. Native electrophoresis permits the separation of ETC complexes into their active forms, and these complexes can then be studied using immunoblotting, in-gel assays (IGA), and purification by electroelution. By combining the results of native gel PAGE with those of other mitochondrial assays, it is possible to obtain a completer picture of ETC activity, its dynamic assembly and disassembly, and how this regulates mitochondrial structure and function. This work will also discuss limitations of these techniques. In summary, the technique of native PAGE, followed by immunoblotting, IGA, and electroelution, presented below, is a powerful way to investigate the functionality and composition of mitochondrial ETC supercomplexes.

This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes.

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in solution, is a relative technique that relies on the elution volume of the analyte to estimate molecular weight. When the protein is not globular or undergoes non-ideal column interactions, the calibration curve based on protein standards is invalid, and the molecular weight determined from elution volume is incorrect. Multi-angle light scattering (MALS) is an absolute technique that determines the molecular weight of an analyte in solution from basic physical equations. The combination of SEC for separation with MALS for analysis constitutes a versatile, reliable means for characterizing solutions of one or more protein species including monomers, native oligomers or aggregates, and heterocomplexes. Since the measurement is performed at each elution volume, SEC-MALS can determine if an eluting peak is homogeneous or heterogeneous and distinguish between a fixed molecular weight distribution versus dynamic equilibrium. Analysis of modified proteins such as glycoproteins or lipoproteins, or conjugates such as detergent-solubilized membrane proteins, is also possible. Hence, SEC-MALS is a critical tool for the protein chemist who must confirm the biophysical properties and solution behavior of molecules produced for biological or biotechnological research. This protocol for SEC-MALS analyzes the molecular weight and size of pure protein monomers and aggregates. The data acquired serve as a foundation for further SEC-MALS analyses including those of complexes, glycoproteins and surfactant-bound membrane proteins.

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering SEC-MALS

This protocol describes the combination of size exclusion chromatography with multi-angle light scattering (SEC-MALS) for absolute characterization of proteins and complexes in solution. SEC-MALS determines the molecular weight and size of pure proteins, native oligomers, heterocomplexes and modified proteins such as glycoproteins.

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in ...

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 ...

Biology

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

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.

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

JoVE Encyclopedia of Experiments

Biological Techniques

Electrophoresis Techniques

Two-Dimensional Polyacrylamide Gel Electrophoresis: A Separation Technique for Analysis of Proteins in Complex Protein Mixture Based on Charge and Molecular Weight

Source: Zhan, X., et al. <a href="https://www.jove.com/v/56739">Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome</a> J. Vis. Exp. (2018)
In this video, we demonstrate a two-dimensional gel electrophoresis method that separates individual proteins from a complex protein mixture obtained from tumor tissues of human nonfunctional pituitary adenoma (NFPA). The proteins are separated based on their electric charge in the first dimension and further separated based on molecular weights in the second dimension.

Source: Zhan, X., et al. Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue ...