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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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 border="0" cellpadding="0" cellspacing="0" width="782">
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:244px;">Slide-A-Lyzer MINI Dialysis Units, 10K MWCO, 0.5 mL</td>
			<td style="width:140px;">Thermo Fisher Scientific</td>
			<td style="width:131px;">69570</td>
			<td style="width:268px;">Pre-equilibrate in dialysis buffer ( if Glycerol removal is desired) 
			Can be self-made according to Fiala et al. 2011</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:244px;">4&#8211;15% Mini-PROTEAN TGX Precast Protein Gels, 10-well,</td>
			<td style="width:140px;">Bio-Rad</td>
			<td style="width:131px;">456-1083</td>
			<td style="width:268px;">Pre-run in running buffer to adjust buffer system</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:244px;">cOmplete, Mini, EDTA-free</td>
			<td style="width:140px;">Roche&#160;</td>
			<td style="width:131px;">11836170001</td>
			<td>use 1 tablet per 50 ml</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:244px;">PBS, pH 7.4&#160; bottle a 500ml Gibco</td>
			<td style="width:140px;">Thermo Fisher Scientific</td>
			<td style="width:131px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:244px;">Ponceau S solution</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:131px;">P7170</td>
			<td style="width:268px;">toxic! wear gloves and protect eyes!</td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:244px;">NativeMark Unstained Protein Standard&#160; 50ul</td>
			<td style="width:140px;">Invitrogen</td>
			<td style="width:131px;">P/N 57030</td>
			<td>load 5 ul/well</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:244px;">A549 cells</td>
			<td style="width:140px;">ATCC</td>
			<td style="width:131px;">ATCC CCL185</td>
			<td>Grow in growth medium (see Table 1)</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:244px;">Vero cells</td>
			<td style="width:140px;">ATCC</td>
			<td style="width:131px;">ATCC CCL81</td>
			<td>Grow in growth medium (see Table 1)</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:244px;">anti-Mx1 antibody</td>
			<td style="width:140px;">Novus Biologicals</td>
			<td style="width:131px;">H00004599_D01P</td>
			<td>Use at a 1:1000 dilution</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:244px;">ECL Anti-rabbit IgG, Horseradish Peroxidase linked whole antibody (from donkey)</td>
			<td style="width:140px;">GE-Healthcare</td>
			<td style="width:131px;">NA934V</td>
			<td>Use at a 1:10000 dilution</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:244px;">0.5% Trypsin-EDTA (1x)&#160;&#160;&#160;&#160;&#160;&#160;&#160; Life Technologies</td>
			<td style="width:140px;">Thermo Fisher</td>
			<td style="width:131px;">15400-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Iodoacetamide&#160;&#160; 5g</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:131px;">I-6125</td>
			<td>stock&#160; 100mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Bromphenolblue</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td>B0126-25G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">DMEM +4.5g/l Gluc,+L-Glut,+Pyruvate life technologies</td>
			<td style="width:140px;">Thermo Fisher Scientific</td>
			<td style="width:131px;">41966-029</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Pen&#160; Strep 100 x&#160;&#160;&#160;&#160; 100ml&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; life technologies</td>
			<td style="width:140px;">Thermo Fisher Scientific</td>
			<td style="width:131px;">15140 - 130</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Glutamax 100xStock, 100ml&#160;&#160;&#160;&#160; life technologies</td>
			<td style="width:140px;">Thermo Fisher Scientific</td>
			<td style="width:131px;">350500-038</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Fetal Bovine Serum, Dialyzed , US Origin 500ml Gibco Lot:42G9552K</td>
			<td style="width:140px;">Thermo Fisher Scientific</td>
			<td style="width:131px;">10270-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Cellulose filter paper</td>
			<td style="width:140px;">Bio-Rad</td>
			<td style="width:131px;">1703965</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">PVDV blotting&#160; membrane</td>
			<td style="width:140px;">GE-Healthcare</td>
			<td style="width:131px;">10600022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Tris(hydroxymethyl)aminomethane</td>
			<td style="width:140px;">Biosolve</td>
			<td style="width:131px;">0020092391BS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">sodium fluoride (NaF)</td>
			<td style="width:140px;">Sigma Aldrich</td>
			<td style="width:131px;">S-7920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">NP-40</td>
			<td style="width:140px;">Calbiochem</td>
			<td style="width:131px;">492015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">cOmplete, Mini, EDTA-free</td>
			<td style="width:140px;">Roche&#160;</td>
			<td style="width:131px;">11836170001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Tween 20</td>
			<td style="width:140px;">Calbiochem</td>
			<td style="width:131px;">6555204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">CHAPS 10% solution</td>
			<td style="width:140px;">Amresco</td>
			<td style="width:131px;">N907</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">DL-Dithiothreitol (DTT)</td>
			<td style="width:140px;">Sigma Aldrich</td>
			<td style="width:131px;">43819</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Glycine</td>
			<td style="width:140px;">Biosolve</td>
			<td style="width:131px;">0007132391BS</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:244px;">sodium orthovanadate (Na3VO4)</td>
			<td style="width:140px;">Sigma Aldrich</td>
			<td style="width:131px;">450243</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Glycerol</td>
			<td style="width:140px;">Sigma Aldrich</td>
			<td style="width:131px;">G7757</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">b-Glycerophospate</td>
			<td style="width:140px;">Sigma Aldrich</td>
			<td style="width:131px;">G9422</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Milk powder</td>
			<td style="width:140px;">Migros/Switzerland</td>
			<td style="width:131px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Methanol</td>
			<td style="width:140px;">Millipore</td>
			<td style="width:131px;">1.06009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">sodium cloride (NaCl)</td>
			<td style="width:140px;">Sigma Aldrich</td>
			<td style="width:131px;">71380</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:244px;">magnesium chloride (MgCl2)</td>
			<td style="width:140px;">Amresco</td>
			<td style="width:131px;">288</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Sodium dodecyl sulphate (SDS)</td>
			<td style="width:140px;">Sigma Aldrich</td>
			<td style="width:131px;">L4509</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">sodium hydroxide (NaOH)</td>
			<td style="width:140px;">Sigma Aldrich</td>
			<td style="width:131px;">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 ...

characterization-multi-subunit-protein-complexes-human-mxa-using-non

Research

JoVE Journal

Biochemistry

10.1K 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

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.
The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

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 ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of RNA-protein interactions. These interactions have been traditionally analyzed with polyacrylamide gels generated between two glass plates and samples electrophoresed vertically. However, polyacrylamide gels cast in trays and electrophoresed horizontally offers several advantages. For example, horizontal gels used to analyze complexes between fluorescent RNA substrates and specific proteins can be imaged multiple times as electrophoresis progresses. This provides the unique opportunity to monitor RNA-protein complexes at several points during the experiment. In addition, horizontal gel electrophoresis makes it possible to analyze many samples in parallel. This can greatly facilitate time course experiments as well as analyzing multiple reactions simultaneously to compare different components and conditions. Here we provide a detailed protocol for generating and using horizontal native gel electrophoresis for analyzing RNA-Protein interactions.

Native polyacrylamide gel electrophoresis is a fundamental tool for analyzing RNA-protein interactions. Traditionally most experiments have used vertical gels. However, horizontal gels provide several advantages, such as the opportunity to monitor complexes during electrophoresis. We provide a detailed protocol for generating and using horizontal native gel electrophoresis.

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis can be characterized using an in vitro DNA synthesis assay followed by polyacrylamide gel electrophoresis. The goal of this assay is to quantify synthesis of both natural DNA and modified DNA (M-DNA). These approaches are particularly useful for resolving oligonucleotides with single nucleotide resolution, enabling observation of individual steps during enzymatic oligonucleotide synthesis. These methods have been applied to the evaluation of an array of biochemical and biophysical properties such as the measurement of steady-state rate constants of individual steps of DNA synthesis, the error rate of DNA synthesis, and DNA binding affinity. By using modified components including, but not limited to, modified nucleoside triphosphates (NTP), M-DNA, and/or mutant DNA polymerases, the relative utility of substrate-DNA polymerase pairs can be effectively evaluated. Here, we detail the assay itself, including the changes that must be made to accommodate nontraditional primer DNA labeling strategies such as near-infrared fluorescently labeled DNA. Additionally, we have detailed crucial technical steps for acrylamide gel pouring and running, which can often be technically challenging.

This protocol describes the characterization of DNA polymerase synthesis of modified DNA through observation of changes to near-infrared fluorescently labeled DNA using gel electrophoresis and gel imaging. Acrylamide gels are used for high resolution imaging of the separation of short nucleic acids, which migrate at different rates depending on size.

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. The ability to readily detect the formation of these fibers under various experimental conditions is essential for understanding their potential function. Many methods have been used to detect the fibers, but not without some drawbacks. For example, electron microscopy (EM), or staining with Congo Red or Thioflavin T often requires purification of the fibers. On the other hand, semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) allows detection of the SDS-resistant amyloid-like fibers in the cell extracts without purification. In addition, it allows the comparison of the size difference of the fibers. More importantly, it can be used to identify specific proteins within the fibers by Western blotting. It is less time consuming and more easily accessible to a wider number of labs. SDD-AGE results often show variable degree of heterogeneity. It raises the question whether part of the heterogeneity results from the dissociation of the protein complex during the electrophoresis in the presence of SDS. For this reason, we have employed a second dimension of SDD-AGE to determine if the size heterogeneity seen in SDD-AGE is truly a result of fiber heterogeneity in vivo and not a result of either degradation or dissociation of some of the proteins during electrophoresis. This method allows fast, qualitative confirmation that the amyloid or amyloid-like fibers are not partially dissociating during the SDD-AGE process.

Here we use two-dimensional semi-denaturing agarose gel electrophoresis to confirm the presence of amyloid-like fibers of heterogeneous size and exclude the possibility that the size heterogeneity is due to dissociation of the amyloid fibers during the gel running process.

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. ...

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

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

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

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

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

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 ...