Name: Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis (Video) | JoVE Journal
Uploaded: 2016-10-28T15:00:00.000+00:00
Duration: 8 min 55 s
Description: Learn to characterize MxA protein oligomeric states using non-denaturing PAGE and western blot analysis with human cell lysates. This accessible method enables researchers to study multi-subunit complexes without purification.

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5 minutes

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

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...

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 ...

The overall goal of this experiment is to determine the number of subunits in homooligomeric protein complexes in cell lysates using non-denaturing sodium dodecosulfate-polyacrylamide gel electrophoresis. This method can help answer key questions in the field of cell biology, such as the oligomeric status of proteins involved in fundamental cellular processes. The main advantage of this technique is that it does not require purification of the homooligomeric protein complexes or specialized, expensive equipment.Therefore, it can be performed in most laboratories. This experiment utilizes two types of cells. A549 cells that express endogenous Myxovirus resistance A protein, or MxA;and virocells that overexpress MxA.Seed the A549 cells and virocells into six well dishes with two milliliters of growth medium per well. Incubate the cells overnight in a cell culture incubator. On the following day, harvest the cells.Wash each well with one milliliter of PBS and attach the cells by adding 0.5 milliliters of 0.25%Trypsin-EDTA solution for approximately five minutes at room temperature. As soon as the cells detach from the dish, add 0.5 milliliters of growth medium, and carefully mix by pipetting up and down. Transfer the cells of each well into a two milliliter tube, and pellet them using a table top centrifuge.Carefully remove the supernatant by pipetting without disturbing the cell pellet. Wash the cells by adding one milliliter of ice cold PBS and carefully pipetting the cell suspension up and down. Pellet the cells in a table top centrifuge.Carefully remove the supernatant by pipetting without detaching the cell pellet. Add 200 microliters of ice cold lysis buffer, resuspend the cells by pipetting up and down, and then place on ice. Immediately protect the lysate from light by covering the tubes with tinfoil because the iodoacetamide in the lysis buffer is light-sensitive.Incubate on ice for 30 minutes. The use of iodoacetamide is crucial because iodoacetamide irreversibly protects the thiol group of free cysteines by forming a thioether bond and prevents artificial protein aggregation. Next, remove cell debris by centrifugation in a pre-chilled table top centrifuge.During the centrifugation, prepare dialysis columns with a molecular weight cutoff of 10, 000. Attach the dialysis columns to a float buoy and place them into a beaker filled with pre-chilled dialysis buffer to equilibrate the columns. Do not touch the membrane.Place a magnetic stirrer in a beaker and transfer the beaker into the cold room. Using the magnetic stirrer to ensure gentle stirring, equilibrate for 20 minutes at four degrees Celsius. After 20 minutes, remove the columns from the dialysis buffer and the float buoy.Transfer the cleared lysates into the prepared dialysis columns by pipetting without touching the membrane. Attach the columns to a float buoy. Put them back into the beaker filled with dialysis buffer, and then transfer the beaker to the cold room.Dialyze the lysate for at least four hours at four degrees Celsius while carefully stirring with a magnetic stirrer to remove small metabolites and salts that could interfere with non-denaturing polyacrylamide gel-electrophoresis. When the dialysis is complete, transfer each dialyzed sample to a 1.5 milliliter tube. Remove precipitates by centrifugation in a table top centrifuge.Electrophoresis must be performed immediately after dialysis of the lysates to prevent the dissociation of the oligomeric protein complexes. Fill the inner and outer gel chamber with pre-chilled running buffer. Pre-run the non-denaturing polyacrylamide gel with pre-chilled running buffer at 25 milliamps per gel for 15 minutes at four degrees Celsius.Mix 15 microliters of the prepared lysate with five microliters of 4X sample buffer. Load a native protein standard of choice and 15 microliters of sample on the gel. Run the gel at 25 milliamps for four hours at four degrees Celsius.When the non-denaturing polyacrylamide gel electrophoresis is complete, disassemble the gel and carefully transfer into SDS buffer. Incubate for 10 minutes at room temperature with gentle shaking. Prepare two sponges, four cellulose filter paper sheets, and a blotting membrane per gel and soak them in blotting buffer.Assemble the sandwich as follows from bottom to top. One sponge, two cellulose filter paper sheets, a blotting membrane, the gel, two cellulose filter paper sheets, and one sponge. Put the sandwich into the blotting tank, making sure the membrane faces the plus pole, while the gel faces the minus pole.Fill the blotting tank with pre-chilled blotting buffer. For the best protein transfer results, blot at 90 milliamps overnight at four degrees Celsius. On the following day, disassemble the sandwich.Incubate the membrane in Ponceau S solution for five minutes at room temperature to visualize the protein standard. Destain the membrane by placing it in deionized water and washing it. After transferring the membrane to a tray, mark the bands of the protein standard using a pen.Block the membrane with blocking buffer for at least one hour at room temperature or overnight at four degrees Celsius. Subsequently, the proteins of interest are visualized by immunostaining using a rabbit polyclonal antibody directed against MxA as described in the text protocol. Virocells lacking endogenous MxA were transfected with recombinant wild type MxA and monomeric and dimeric MxA variance.The migration of the recombinant MxA variance at their expected molecular weight confirmed their oligomeric state. The ectopically expressed wild type MxA migrated as a tetramer. These recombinant proteins were then used to assess the oligomeric state of endogenous human MxA protein derived from interferon-alpha stimulated A549 cells.The result revealed that the size of MxA in lysates of interferon-alpha stimulated A549 cells corresponds to a tetramer. Once mastered, this technique can be done in one and a half days if it is performed properly. While attempting this procedure, it's important to remember not to freeze and thaw the samples during the entire procedure, since this could destroy protein interactions or cause protein aggregations.Additionally, when working with light-sensitive reagents, like iodoacetamide, it is important to protect the samples from light during the lysis step. Following the non-denaturing SDS-PAGE, other methods like protein gel extraction and enzymatic activity AsiS or a second dimension PAGE can be performed in order to answer additional questions like enzymatic activity and the characterization of potential hetero-oligomeric complexes. After its development, this technique paved the way for researchers in the field of cell biology to explore the composition of oligomeric protein complexes in cultured cells.After watching this video, you should have a good understanding of how to prepare cell lysates from cell cultures to study oligomeric proteins in non-denaturing PAGE analysis. Don't forget that working with iodoacetamide can be extremely hazardous and precautions such as wearing gloves and eye protection should always be taken.

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

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