Name: horizontal gel electrophoresis for enhanced detection of protein-rna complexes
Uploaded: 2017-07-28T14:00:00
Description: Watch this Scientific Journal Video about Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes at JoVE.com

We thank Laura Vanderploeg for preparing figures. Work in the Sheets lab is supported by NSF grant 1050395 and NIH grant (R21HD076828). Work in the Ryder lab is supported by NIH grants R01GM117237 and R01GM117008. Megan Dowdle is supported by a SciMed GRS Advanced Opportunity Fellowship through University of Wisconsin-Madison Graduate School and Biotechnology Training Program through the National Institute of General Medical Sciences of the National Institutes of Health (T32GM008349).

1. Preparation of the Horizontal Native Polyacrylamide Gel4.
<ol>
	<li>Preparation of the materials needed.
	<ol>
		<li>For the horizontal gel apparatus, use a gel box (37 cm x 24 cm) with a 27 cm x 21 cm tray and a capacity for two 24-well combs. This setup provides a total of 48 samples that can be analyzed simultaneously.</li>
		<li>Prepare the following reagents: 40% Acrylamide-Bis 19:1, 5x TBE (Tris-Borate-EDTA pH 8.0), TEMED, and 10% APS (ammonium persulfate).</li>
	</ol>
...

<ol>
	<li>Ryder, S. P., Recht, M. I., Williamson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+protein-RNA+interactions+by+gel+mobility+shift.">Quantitative analysis of protein-RNA interactions by gel mobility shift.</a> Methods Mol Biol. 488, 99-115 (2008).</li><li>Dahlberg, A. E., Dingman, C. W., Peacock, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+characterization+of+bacterial+polyribosomes+in+agarose-acrylamide+composite+gels.">Electrophoretic characterization of bacterial polyribosomes in agarose-acrylamide composite gels.</a> J Mol Biol. 41 (1), 139-147 (1969).</li><li>Hellman, L. M., Fried, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+mobility+shift+assay+(EMSA)+for+detecting+protein-nucleic+acid+interactions.">Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions.</a> Nat Protoc. 2 (8), 1849-1861 (2007).</li><li>Pagano, J. M., Farley, B. M., Essien, K. I., Ryder, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+recognition+by+the+embryonic+cell+fate+determinant+and+germline+totipotency+factor+MEX-3.">RNA recognition by the embryonic cell fate determinant and germline totipotency factor MEX-3.</a> Proc Natl Acad Sci USA. 106 (48), 20252-20257 (2009).</li><li>Royer, C. A., Scarlata, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+approaches+to+quantifying+biomolecular+interactions.">Fluorescence approaches to quantifying biomolecular interactions.</a> Meth Enzymol. 450, 79-106 (2008).</li><li>Su, C., Wang, F., Ciolek, D., Pan, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoresis+of+proteins+and+protein-protein+complexes+in+native+polyacrylamide+gels+using+a+horizontal+gel+apparatus.">Electrophoresis of proteins and protein-protein complexes in native polyacrylamide gels using a horizontal gel apparatus.</a> Anal Biochem. 223, 93-98 (1994).</li><li>Buczek, P., Horvath, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamic+Characterization+of+Binding+Oxytricha+nova+Single+Strand+Telomere+DNA+with+the+Alpha+Protein+N-terminal+Domain.">Thermodynamic Characterization of Binding Oxytricha nova Single Strand Telomere DNA with the Alpha Protein N-terminal Domain.</a> J Mol Biol. 359 (5), 1217-1234 (2006).</li><li>Zhang, Y., Park, S., Blaser, S., Sheets, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+RNA+binding+and+translational+repression+by+the+Bicaudal-C+regulatory+protein.">Determinants of RNA binding and translational repression by the Bicaudal-C regulatory protein.</a> J Biol Chem. 289 (11), 7497-7504 (2014).</li><li>Gamberi, C., Lasko, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bic-C+family+of+developmental+translational+regulators.">The Bic-C family of developmental translational regulators.</a> Comp Funct Genomics. , 141386 (2012).</li><li>Saffman, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Premature+translation+of+oskar+in+oocytes+lacking+the+RNA-binding+protein+bicaudal-C.">Premature translation of oskar in oocytes lacking the RNA-binding protein bicaudal-C.</a> Mol Cell Biol. 18 (8), 4855-4862 (1998).</li><li>Chicoine, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bicaudal-C+recruits+CCR4-NOT+deadenylase+to+target+mRNAs+and+regulates+oogenesis,+cytoskeletal+organization,+and+its+own+expression.">Bicaudal-C recruits CCR4-NOT deadenylase to target mRNAs and regulates oogenesis, cytoskeletal organization, and its own expression.</a> Dev Cell. 13 (5), 691-704 (2007).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bicaudal-C+spatially+controls+translation+of+vertebrate+maternal+mRNAs.">Bicaudal-C spatially controls translation of vertebrate maternal mRNAs.</a> RNA. 19 (11), 1575-1582 (2013).</li><li>Maisonneuve, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bicaudal+C,+a+novel+regulator+of+Dvl+signaling+abutting+RNA-processing+bodies,+controls+cilia+orientation+and+leftward+flow.">Bicaudal C, a novel regulator of Dvl signaling abutting RNA-processing bodies, controls cilia orientation and leftward flow.</a> Development. 136 (17), 3019-3030 (2009).</li><li>Tran, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNA-binding+protein+bicaudal+C+regulates+polycystin+2+in+the+kidney+by+antagonizing+miR-17+activity.">The RNA-binding protein bicaudal C regulates polycystin 2 in the kidney by antagonizing miR-17 activity.</a> Development. 137 (7), 1107-1116 (2010).</li><li>Pagano, J. M., Clingman, C. C., Ryder, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+approaches+to+monitor+protein-nucleic+acid+interactions+using+fluorescent+probes.">Quantitative approaches to monitor protein-nucleic acid interactions using fluorescent probes.</a> RNA. 17 (1), 14-20 (2011).</li><li>Foley, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platform+to+Enable+the+Pharmacological+Profiling+of+Small+Molecules+in+Gel-Based+Electrophoretic+Mobility+Shift+Assays.">Platform to Enable the Pharmacological Profiling of Small Molecules in Gel-Based Electrophoretic Mobility Shift Assays.</a> J Biomol Screen. 21 (10), 1125-1131 (2016).</li></ol>

Native polyacrylamide gels are an invaluable tool for investigating protein-RNA interactions and traditionally these gels are electrophoresed vertically2,3. We have used a modification of the protocol that substitutes native polyacrylamide gels created and electrophoresed horizontally1,4,6,7,15. These ...

Electrophoretic mobility shift assays (EMSAs) have proven to be an invaluable biochemical tool to analyze specific protein-nucleic acid interactions1,2,3. These assays can provide important information regarding the binding affinity of proteins to RNA or DNA3, the component stoichiometry of nucleic acid-protein complexes1 and provide important new insights about the binding specificity of RNA binding proteins via substrate competition experiments1.
The traditional experimental setup for these assays consists of mixing purified protein with a radiolabeled RNA substrate. The resulting complexes are then analyzed with non-denaturing (native) polyacrylamide gels poured between two glass plates followed by sample electrophoresis in a vertical apparatus3. While this approach has been used exhaustively to provide important insights in the biochemical mechanisms that underlie the binding of proteins to nucleic acids, it also has several limitations. For example, this basic strategy has relatively low throughput and it is not readily adaptable for applications that require analyzing many binding reactions in parallel. In addition, with the traditional vertical apparatus it is challenging to potentially monitor complexes at multiple times during electrophoresis3,4.
Here we present an adaptation of the EMSA assay that uses native polyacrylamide gels cast in a flatbed apparatus, horizontal electrophoresis and fluorescently labeled RNA substrates4,5,6,7. The incorporation of these relatively simple modifications to the basic strategy provides some powerful advantages. In particular, the horizontal flatbed format easily lends itself to analyzing dozens of samples simultaneously4. Also, for some RNA-protein complexes, such as those formed between the Bicaudal-C protein and its RNA substrate electrophoresis in a horizontal gel provides an increased ability to resolve distinct RNA-protein complexes and discriminate these from unbound RNA substrate.

<table border="0" cellpadding="0" cellspacing="0" width="1432">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Horizontal Gel Box</td>
			<td style="width:112px;">OWL</td>
			<td style="width:116px;">N/A</td>
			<td style="width:831px;">Product no longer made. Similar gel boxes can be found at Thermo Scientific, A-series gel boxes. Catalog Number: A2-BP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well large large horizontal gel electrophoresis combs</td>
			<td>OWL</td>
			<td>N/A</td>
			<td>Product no longer made. Similar gel boxes can be found at Thermo Scientific, A-series gel box combs. Catalog Number: A2-24C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Powerpac 300</td>
			<td>Bio-Rad</td>
			<td>1655050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-Protean II Electrophoresis Cell</td>
			<td>Bio-Rad</td>
			<td>165-2940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">InstaPAGE-19 40% 19:1 Acrylamide/Bis</td>
			<td>IBI</td>
			<td>IB70015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TEMED</td>
			<td>IBI</td>
			<td>IB70120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">APS</td>
			<td>IBI</td>
			<td>IB70080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast tRNAs</td>
			<td>Ambion</td>
			<td>AM7119</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescein labeled RNA</td>
			<td>IDT</td>
			<td>N/A</td>
			<td>Order can be made custom to length and desired sequence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA tetrasodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>E5391-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris Base Ultrapure</td>
			<td>US Biological</td>
			<td>T8600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Boric Acid</td>
			<td>Fisher Scientific</td>
			<td>BP168-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP366-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DEPC</td>
			<td>Sigma-Aldrich</td>
			<td>1609-47-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreitol (DTT)</td>
			<td>Sigma-Aldrich</td>
			<td>3483 12 3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

To demonstrate the power and versatility of the horizontal native gel electrophoresis we analyzed the binding of the Xenopus Bicaudal-C (Bicc1) protein to a fluorescently labeled RNA containing a Bicc1 binding site. Bicc1 proteins function as mRNA-specific translational repressors to control cell fate decisions during the maternal stages of animal development9,10,11,<sup class...

Watch this Scientific Journal Video about Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes at JoVE.com

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

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 ...

The overall of this protocol is to analyze RNA protein interactions using horizontal native gel electrophoresis. This method can help answer key questions in RNA biochemistry, such as defining the characteristics of specific RNA protein interactions. The main advantage of this technique is that it allows complex formation to be monitored multiple times during electrophoresis.For the horizontal gel apparatus, use a gel box with a 27 centimeter by 21 centimeter casting tray and a capacity for two 24 well combs. This set up provides a total of 48 samples that can be analyzed simultaneously. Prepare two liters of 1x TBE running buffer by diluting 400 milliliters of 5x TBE with 1, 600 milliliters of water and mixing.Place the TBE buffer in the cold room to cool. Prepare the following reagents following standard procedures, 40%acrylamide best 19:1, 5x TBE, temed and 10%ammonium per sulfate. To make a 10%native acrylamide gel, add the following to a 500 milliliter flask:80 milliliters of 5x TBE, 100 milliliters of 40%acrylamide and water to a final volume of 400 milliliters.Add four milliliters of 10%ammonium per sulfate and 400 microliters of temed and mix well. Pour the mix into the horizontal casting tray and allow the gel to polymerize in the fume hood for 30 minutes. The polymerized gel will be approximately two centimeters thick and a thin layer of liquid will remain on top of the gel.Place the gel into the gel box and take it into the cold room. Next, pour the liquid off and rinse with running buffer. Remove the combs carefully, add two liters of cold running buffer to the gel box and then use a syringe to rinse the wells with running buffer.Pre-run the gel at 120 volts for one hour at four degrees celsius in a cold room. While the gel is being prerun, prepare the binding reactions. Add the following binding reaction components to each RNA free microcentrifuge tube in this order:five microliters of 10 millimolar BSA, five microliters of 20 millimolar DTT, five microliters of 10 millimolar yeast tRNAs, 10 microliters of 5x binding buffer, bicaudal-c protein to a final concentration of 250 nanomolar in 50 microliters, DEPC-treated water to a final volume of 45 microliters and five microliters of fluorescently labeled RNA substrate.Close the tubes and flick gently to mix, centrifuge the tubes briefly and incubate in the dark for 30 minutes at room temperature. When the binding reactions are complete, add 15 microliters of 50%glycerol to each reaction and mix gently. Carefully load 30 microliters of each reaction into the wells of the gel, the amount of sample that can be loaded into a single well varies depending on the thickness of the gel and the size of the wells.The power supply to the gel apparatus, switch on the power, adjust the voltage to 120 volts and run the gel in four degree celsius. To prevent damaging or bleaching of the fluorescently labeled RNA, conduct electrophoresis in the dark. Electrophoresis times will vary depending upon the specifics of the RNA protein complex being analyzed.A major advantage of the horizontal electrophorated mobility shift assay analysis, is that electrophoresis can be stopped in the gel image prior to completion of the run. Perform analysis with any instrument designed for detecting an imaging fluorescent substrates. Adjust the imager settings, for fluorescently labeled ligands, use the following settings.Fluorescein wavelength of 473 nanometers, photomultiplier voltage of 750 volts and pixel size of 100 micrometers. Scan the gel to capture the image. After imaging, the gel can be placed back into the apparatus and electrophoresis can be continued for longer times.The binding of the xenopus bicaudal-C or Bicc1 protein to a fluorescently labeled Cripto1 mRNA containing a Bicc1 binding site, was analyzed by both vertical and horizontal native gel electrophoresis, as a negative control, Bicc1 was mixed with a fluorescently labeled RNA derived from CyclinB1 mRNA. It was readily apparent using either method, that Bicc1 binds to the Cripto1 RNA and forms a specific complex. However, the Bicc1, Cripto1 complexes were significantly more distinct when analyzed with the horizontal gel, facilitating their detection and allowing the discrimination of protein RNA complexes from unbound RNA.After imaging, the gel was returned to the gel apparatus, electrophoresis for an additional three and a half hours, and reimaged. As shown here, the complexes had migrated further and were still readily detectable, indicating their stability. Once mastered, this technique can be done in four to five hours, if it is performed properly.After watching this video, you should have a good understanding of how to analyze RNA protein interactions using horizontal native gel electrophoresis.

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Research

JoVE Journal

Biochemistry

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

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

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

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

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.

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 ...

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 ...

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

We demonstrate methods for the expression and purification of tRNA(UUU) in Escherichia coli and the analysis by gel retardation assays of the binding of tRNA(UUU) to TcdA, an N6-threonylcarbamoyladenosine (t6A) dehydratase, which cyclizes the threonylcarbamoyl side chain attached to A37 in the anticodon stem loop (ASL) of tRNAs to cyclic t6A (ct6A). Transcription of the synthetic gene encoding tRNA(UUU) is induced in E. coli with 1 mM isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) and the cells containing tRNA are harvested 24 h post-induction. The RNA fraction is purified using the acid phenol extraction method. Pure tRNA is obtained by a gel filtration chromatography that efficiently separates the small-sized tRNA molecules from larger intact or fragmented nucleic acids. To analyze TcdA binding to tRNA(UUU), TcdA is mixed with tRNA(UUU) and separated on a native agarose gel at 4 &#176;C. The free tRNA(UUU) migrates faster, while the TcdA-tRNA(UUU) complexes undergo a mobility retardation that can be observed upon staining of the gel. We demonstrate that TcdA is a tRNA(UUU)-binding enzyme. This gel retardation assay can be used to study TcdA mutants and the effects of additives and other proteins on binding.

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

A protocol is presented for the production of tRNA(UUU) and the analysis of tRNA(UUU) in complex with the enzyme TcdA by agarose gel retardation assays.

We demonstrate methods for the expression and purification of tRNA(UUU) in Escherichia coli and the analysis by gel retardation assays of the binding of ...

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 ...

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 ...

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. ...

Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve classes of intrinsically disordered proteins (IDPs) to reduce oxidative and osmotic stress. This article uses a combination of capillary gel electrophoresis (CGE) and mobility shift affinity electrophoresis (ACE) in order to describe the binding behavior of different conformers of the IDP AtHIRD11 from Arabidopsis thaliana. CGE is used to confirm the purity of AtHIRD11 and to exclude fragments, posttranslational modifications, and other impurities as reasons for complex peak patterns. In this part of the experiment, the different sample components are separated by a viscous gel inside a capillary by their different masses and detected with a diode array detector. Afterward, the binding behavior of the sample towards various metal ions is investigated by ACE. In this case, the ligand is added to the buffer solution and the shift in migration time is measured in order to determine whether a binding event has occurred or not. One of the advantages of using the combination of CGE and ACE to determine the binding behavior of an IDP is the possibility to automate the gel electrophoresis and the binding assay. Furthermore, CGE shows a lower limit of detection than the classical gel electrophoresis and ACE is able to determine the manner of binding a ligand in a fast manner. In addition, ACE can also be applied to other charged species than metal ions. However, the use of this method for binding experiments is limited in its ability to determine the number of binding sites. Nevertheless, the combination of CGE and ACE can be adapted for characterizing the binding behavior of any protein sample towards numerous charged ligands.

This protocol combines the characterization of a protein sample by capillary gel electrophoresis and a fast-binding screening for charged ligands by affinity capillary electrophoresis. It is recommended for proteins with a flexible structure, such as intrinsically disordered proteins, to determine any differences in binding for different conformers.

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve ...

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 ...