Name: identification of myod interactome using tandem affinity purification coupled to mass spectrometry
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Description: Watch this Scientific Journal Video about Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry at JoVE.com

Work in the Ait-Si-Ali lab was supported by the Association Fran&#231;aise contre les Myopathies T&#233;l&#233;thon (AFM-T&#233;l&#233;thon); Institut National du Cancer (INCa); Agence Nationale de la Recherche (ANR), Fondation Association pour la Recherche sur le Cancer (Fondation ARC); Groupement des Entreprises Fran&#231;aises pour la Lutte contre le Cancer (GEFLUC); CNRS; Universit&#233; Paris Diderot and the ''Who Am I?'' Laboratory of Excellence #ANR-11- LABX-0071 funded by the French Government through its ''Investments for the Future'' program operated by the ANR under grant #ANR-11-IDEX-0005-01. EB was supported by an INCa grant.

1. Preparation of HeLa-S3 Nuclear Salt-extractable and Chromatin-bound Fractions
<ol>
	<li>Collection of the Cells
	<ol>
		<li>Grow HeLa-S3 Flag-HA-MyoD and HeLa-S3 Flag-HA (control cell line) in Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 &#181;g/ml streptomycin (growth medium) at 37 &#176;C and 5% CO2 in a humid incubator. 
		Note: HeLa-S3 cells stably expressing Flag-HA-MyoD and HeLa-S3 cells transduced with the empty vector (HeLa-S3...

<ol>
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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+class+II+histone+deacetylases+with+heterochromatin+protein+1:+potential+role+for+histone+methylation+in+control+of+muscle+differentiation.">Association of class II histone deacetylases with heterochromatin protein 1: potential role for histone methylation in control of muscle differentiation.</a> Mol Cell Biol. 22 (20), 7302-7312 (2002).</li><li>Forcales, S. 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The presented method permits exhaustive identification of partners of a transcription factor, MyoD. It revealed MyoD partners in a heterologous system resistant to MyoD-induced differentiation, namely HeLa-S3 cells. Thus, by definition, identified MyoD partners are ubiquitously expressed. These include general and sequence-specific transcription factors, chromatin-modifying enzymes, RNA processing proteins, kinases (Tables 1 and 2). Since HeLa-S3 cells are resistant to MyoD-induced trans...

Eukaryotic multi-cellular organisms are composed of different organs and tissues. Each functional tissue has a specific gene pattern expression, which is determined at each differentiation step. Cellular differentiation involves activation of specific genes, maintenance of their expression and, generally, silencing of a set of genes such those involved in cell proliferation. Skeletal muscle differentiation, or myogenesis, is thus a multi-step process, that begins with the determination of mesodermal stem cells into myoblasts, and then leads to the terminal differentiation of these myoblasts into first mono-nucleated, and then multi-nucleated, myotubes. Thus, myoblasts are &#34;determined&#34; cells, that are still able to proliferate, but they are committed to the skeletal muscle lineage, and thus can differentiate solely into skeletal muscle cells either during embryonic development or in adult muscle regeneration. The process of skeletal muscle terminal differentiation is orchestrated by a specific genetic program that begins with the permanent exit from the cell cycle of myoblast precursor cells that leads to a definitive silencing of proliferation associated genes, such as E2F target genes1. Indeed, during the process of terminal differentiation, myoblast proliferation arrest is a crucial step that precedes the expression of skeletal muscle specific genes and the fusion of myoblasts into myotubes2. Such a program permits adult muscle stem cells, also called satellite cells, to differentiate during the regeneration process following skeletal muscle injury.
Mammalian myogenesis is critically dependent on a family of myogenic basic helix-loop-helix (bHLH) transcription factors MyoD, Myf5, MRF4 and Myogenin, frequently referred to as the family of skeletal muscle determination factors or MRFs (Muscle Regulatory Factors)3. Each of them plays an essential role in specification and differentiation of skeletal muscle cells and has a specific expression pattern45-7. The activation of Myf5 and MyoD constitutes the determinative step that commits cells to the myogenic lineage, and subsequent expression of Myogenin triggers myogenesis with activation of skeletal muscle specific genes, such as MCK (Muscle Creatine Kinase). Myogenic bHLH transcription factors cooperate with members of the MEF2 family in the activation of muscle genes from previously silent loci8. They also stimulate skeletal muscle gene transcription as heterodimers with ubiquitous bHLH proteins, E12 and E47, known as E proteins, which bind so-called E-boxes in various gene-regulatory regions8. Twist, Id (inhibitor of differentiation) and other factors negatively regulate this process, by competing with MyoD for E proteins binding8.
MyoD is considered as the major player in triggering muscle terminal differentiation9 since it has the capacity to induce a myogenic determination/differentiation (trans-differentiation) program in many fully differentiated non-muscle systems10-13. Indeed, forced expression of MyoD induces the trans-differentiation of different cellular types even those derived from another embryologic origin12. For example, MyoD can convert hepatocytes, fibroblasts, melanocytes, neuroblasts, and adipocytes into muscle-like cells. The trans-differentiative action of MyoD involves an abnormal activation of the myogenic genetic program (notably its target genes) in a non-muscular environment, concomitant to the silencing of the original genetic program (notably, proliferation genes).
In proliferating myoblasts, MyoD is expressed but is unable to activate its target genes even when it binds to their promoters14-16. Therefore, the requirement for MyoD to be continuously expressed in undifferentiated myoblasts remains quite elusive. MyoD could repress its target genes due to recruitment of repressive chromatin-modifying enzymes in proliferating myoblasts prior to loading of activating chromatin remodeling enzymes14,17. For example, in proliferating myoblasts, MyoD is associated with transcriptional co-repressor KAP-1, histone deacetylases (HDACs) and repressive lysine methyltransferases (KMTs), including histone H3 lysine 9 or H3K9 and H3K27 KMTs, and actively suppress its target genes expression by establishing a locally repressive chromatin structure14,17. Importantly, a recent report indicated that MyoD is itself directly methylated by the H3K9 KMT G9a resulting in inhibition of its transactivating activity16.
The epigenetic mechanisms involved in this trans-differentiation of non-muscle cells by MyoD are largely unknown. Notably, some cell lines are resistant to MyoD-induced trans-differentiation. Thus, in HeLa cells, MyoD is either inactive or even might function as repressor rather than activator of transcription due to lack of expression of the BAF60C subunit of the chromatin remodeling complex SWI/SNF18. This model can thus be of choice to better characterize the mechanisms of MyoD-induced gene repression. It is also suitable to assay the ability of MyoD to induce repressive chromatin environment at its target loci with its associated partners and therefore uncover the MyoD-dependent repressive mechanisms in proliferating myoblasts to fine-tune terminal differentiation.
Here we describe the protocol for the identification of MyoD partners by using Tandem Affinity Purification (TAP-Tag) coupled to mass spectrometry (MS) characterization. The use of HeLa-S3 cells stably expressing Flag-HA-MyoD permitted to get enough material to purify the MyoD complexes from fractionated nuclear extracts. The identification of MyoD partners in the heterologous system was followed by validation in a relevant system.

<table border="0" cellpadding="0" cellspacing="0" width="543">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Cell lines</td>
			<td style="width:115px;"></td>
			<td style="width:101px;"></td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">HeLa-S3</td>
			<td style="width:115px;">ATCC</td>
			<td style="width:101px;">CCL-2.2</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">C2C12</td>
			<td style="width:115px;">ATCC</td>
			<td style="width:101px;">CRL-1772</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Name</td>
			<td style="width:115px;">Company</td>
			<td style="width:101px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Equipment</td>
			<td style="width:115px;"></td>
			<td style="width:101px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Spinner</td>
			<td style="width:115px;">Corning</td>
			<td style="width:101px;">778531</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:177px;">Homogenizer :&#160; Dounce homogenizer&#160;</td>
			<td style="width:115px;">Wheaton</td>
			<td style="width:101px;">432-1273 and 432-1271</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Agitating device for spinners</td>
			<td style="width:115px;">Bellco</td>
			<td style="width:101px;">778531</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Sonicator</td>
			<td style="width:115px;">Diagenode</td>
			<td style="width:101px;">UCD 200</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Hemocytometer</td>
			<td style="width:115px;">Marienfeld Superior&#160;</td>
			<td style="width:101px;">640610</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Low-binding tubes</td>
			<td style="width:115px;">Sorenson</td>
			<td style="width:101px;">27210</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Empty spin column</td>
			<td style="width:115px;">Bio-Rad</td>
			<td style="width:101px;">7326204</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Name</td>
			<td style="width:115px;">Company</td>
			<td style="width:101px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Reagents</td>
			<td style="width:115px;"></td>
			<td style="width:101px;"></td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:177px;">SDS-polyacrylamide 4-12 %&#160; gel</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:101px;">NP0336BOX</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;">4X loading buffer&#160;</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:101px;">NP0007</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;">10X reducing agent</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:101px;">NP0004</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Silver staining kit</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:101px;">A8592</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Centrifugal filter units, 10K</td>
			<td style="width:115px;">Millipore</td>
			<td style="width:101px;">UFC501024</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Protein G agarose beads</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">P4691</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Protein A/G&#160; Resin</td>
			<td style="width:115px;">Thermo Scientific</td>
			<td style="width:101px;">53132</td>
			<td></td>
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		<tr height="34">
			<td height="34" style="height:34px;width:177px;">Flag resin (anti-Flag M2-agarose affinity gel)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">A2220</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:177px;">HA resin (monoclonal Anti-HA agarose)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">A2095</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">MNase</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">N3755</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:177px;">Flag free peptide (DYKDDDDK)</td>
			<td style="width:115px;">Ansynth Service BV</td>
			<td style="width:101px;">Custom synthesis</td>
			<td>Resuspend up to 4 mg/mL in 50 mM Tris-HCl, pH 7.8</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:177px;">HA free peptide (YPYDVPDYA)</td>
			<td style="width:115px;">Ansynth Service BV</td>
			<td style="width:101px;">Custom synthesis</td>
			<td>Resuspend up to 4 mg/mL in 50 mM Tris-HCl, pH 7.8</td>
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		<tr height="34">
			<td height="34" style="height:34px;width:177px;">Bicinchoninic acid based protein assay kit : BCA kit</td>
			<td style="width:115px;">Thermo Scientific</td>
			<td style="width:101px;">23225</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Protease inhibitors</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">S8830</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:177px;">Luminol-based enhanced chemiluminescence (ECL) HRP substrate</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:101px;">34075</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">BSA</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">A9647</td>
			<td></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;">Sheared salmon sperm DNA (Deoxyribonucleic acid sodium salt from salmon testes)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">D1626</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;">Spermine tetrahydrochloride</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">S1141</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;">Spermidine</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">S0266</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;">Glycerol</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">G5516</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PBS</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">D8537</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MOPS running buffer</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:101px;">NP0001</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DMEM (high glucose)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">D0822</td>
			<td>Pre-warm&#160; at 37 &#176;C before use</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Trypsin-EDTA (0.05% phenol red</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:101px;">25300-054</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;">Serum</td>
			<td>GE healthcare life sciences&#160;</td>
			<td style="width:101px;">PAA A15-102</td>
			<td style="width:151px;">Each lot of serum has to be first tested for your cells.</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Penicillin and Streptomycin&#160;</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:101px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:101px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Water (sterile-filtered)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">W3500</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Name</td>
			<td style="width:115px;">Company</td>
			<td style="width:101px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Antibodies</td>
			<td style="width:115px;"></td>
			<td style="width:101px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">HA from rat (12CA5)</td>
			<td style="width:115px;">Roche</td>
			<td style="width:101px;">11583816001</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Flag</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">A8592</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">MyoD</td>
			<td style="width:115px;">Santa Cruz</td>
			<td>sc-32758</td>
			<td>To use for western blotting</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">MyoD</td>
			<td style="width:115px;">Santa Cruz</td>
			<td>SC-760</td>
			<td>To use for immunoprecipitation and western blotting</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">CBFbeta</td>
			<td style="width:115px;">Santa Cruz</td>
			<td style="width:101px;">FL-182&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">HP1alpha</td>
			<td style="width:115px;">Euromedex</td>
			<td style="width:101px;">2HP2G9</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">HP1beta</td>
			<td style="width:115px;">Euromedex</td>
			<td style="width:101px;">1MOD1A9AS</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">HP1gamma</td>
			<td style="width:115px;">Euromedex</td>
			<td style="width:101px;">2MOD1GC</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:177px;">Suv39h1</td>
			<td style="width:115px;">Cell Signaling Technology</td>
			<td style="width:101px;">8729</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">BAF47</td>
			<td style="width:115px;">BD Biosciences</td>
			<td style="width:101px;">612111</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Myf5</td>
			<td style="width:115px;">Santa Cruz</td>
			<td>SC-302</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Tubulin</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">T9026</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Actin</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">A5441</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">IgG Mouse</td>
			<td style="width:115px;">Santa Cruz</td>
			<td style="width:101px;">SC-2025</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">IgG Rabbit</td>
			<td style="width:115px;">Santa Cruz</td>
			<td style="width:101px;">SC-2027</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Goat anti-rat -HRP</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">A9037</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Goat anti-rabbit -HRP</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">A6154&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Goat anti-mouse -HRP</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">A4416</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of myod interactome using tandem affinity purification coupled to mass spectrometry

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

To understand the regulation of MyoD activity, we undertook the exhaustive characterization of the MyoD complexes using biochemical purification, based on the immunopurification of a double tagged form of MyoD followed by mass spectrometry (MS). The use of HeLa-S3 cell line expressing Flag-HA-tagged MyoD and a control cell line expressing Flag-HA permits to get enough material to purify the MyoD complex by performing double-affinity purification of Flag-HA-MyoD.
<p class="jove_content...

Watch this Scientific Journal Video about Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry at JoVE.com

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...

Research

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Biology

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