Name: dynamic proteomic and mirna analysis of polysomes from isolated mouse heart after langendorff perfusion
Uploaded: 2018-08-29T13:00:00
Description: Watch this Scientific Journal Video about Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion at JoVE.com

HL112730 NIH P01 (trapo, JVE), NIH R01 HL132075 (trapo, JVE), centro del corazón de la mujer de Barbra Streisand (trapo, JVE), Dorothy y Phillip Lyon E. Sillón en Cardiología Molecular (GAR), Erika Glazer dotado Cátedra salud del corazón de la mujer (JVE) y la Academia Checa de Ciencias Apoyo institucional RVO: 68081715 (MS).

Todos los estudios en animales fueron realizados de acuerdo con las directrices y aprobados por el cuidado de Animal institucional y Comité uso de Cedars-Sinai Medical Center.
1. Langendorff perfusión del corazón de ratón
<ol><li>
Langendorff perfusión del corazón de ratón con isquemia y reperfusión
	<ol>
<li>Administración intraperitoneal pentobarbital de sodio 70 mg/kg para el ratón adulto (8 semanas de edad, varón, C57BL6/j). Confirmar la anestesia profunda por...

<ol>
	<li>Pourpirali, S., Valacca, C., Merlo, P., Rizza, S., D&#8217;Amico, S., Cecconi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+pseudohypoxia+targets+ambra1+mrna+to+p-bodies+for+translational+repression.">Prolonged pseudohypoxia targets ambra1 mrna to p-bodies for translational repression.</a> PLoS ONE. 10, e0129750 (2015).</li><li>Andres, A. M., Tucker, K. C., Thomas, A., Taylor, D. J., Sengstock, D., Jahania, S. M., Dabir, R., Pourpirali, S., Brown, J. A., Westbrook, D. G., Ballinger, S. W., Mentzer, R. M., Gottlieb, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy+and+mitochondrial+biogenesis+in+atrial+tissue+of+patients+undergoing+heart+surgery+with+cardiopulmonary+bypass.">Mitophagy and mitochondrial biogenesis in atrial tissue of patients undergoing heart surgery with cardiopulmonary bypass.</a> JCI Insight. 2, e89303 (2017).</li><li>Kislinger, T., Cox, B., Kannan, A., Chung, C., Hu, P., Ignatchenko, A., Scott, M. S., Gramolini, A. O., Morris, Q., Hallett, M. T., Rossant, J., Hughes, T. R., Frey, B., Emili, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+survey+of+organ+and+organelle+protein+expression+in+mouse:+Combined+proteomic+and+transcriptomic+profiling.">Global survey of organ and organelle protein expression in mouse: Combined proteomic and transcriptomic profiling.</a> Cell. 125, 173-186 (2006).</li><li>Kren, B. T., Wong, P. Y., Shiota, A., Zhang, X., Zeng, Y., Steer, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+trafficking+of+transcripts+and+micrornas+in+regenerating+liver+after+partial+hepatectomy.">Polysome trafficking of transcripts and micrornas in regenerating liver after partial hepatectomy.</a> American Journal of Physiology. Gastrointestinal and Liver Physiology. 297, G1181-G1192 (2009).</li><li>Gottlieb, R. A., Pourpirali, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lost+in+translation:+Mirnas+and+mrnas+in+ischemic+preconditioning+and+ischemia/reperfusion+injury.">Lost in translation: Mirnas and mrnas in ischemic preconditioning and ischemia/reperfusion injury.</a> Journal of Molecular and Cellular Cardiology. 95, 70-77 (2016).</li><li>Coudert, L., Adjibade, P., Mazroui, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+translation+initiation+during+stress+conditions+by+polysome+profiling.">Analysis of translation initiation during stress conditions by polysome profiling.</a> Journal of Visualized Experiments. , (2014).</li><li>Lorsch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+enzymology.+Laboratory+methods+in+enzymology:+Rna.+Preface.">Methods in enzymology. Laboratory methods in enzymology: Rna. Preface.</a> Methods in Enzymology. 530, xxi (2013).</li><li>Kuzniewska, B., Chojnacka, M., Milek, J., Dziembowska, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+polysomal+fractions+from+mouse+brain+synaptoneurosomes+and+analysis+of+polysomal-bound+mrnas.">Preparation of polysomal fractions from mouse brain synaptoneurosomes and analysis of polysomal-bound mrnas.</a> Journal of Neuroscience Methods. 293, 226-233 (2018).</li><li>He, S. L., Green, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+analysis+of+mammalian+cells.">Polysome analysis of mammalian cells.</a> Methods in Enzymology. 530, 183-192 (2013).</li><li>Molotski, N., Soen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+association+of+micrornas+with+polysomes+reflects+distinct+strengths+of+interactions+with+their+mrna+targets.">Differential association of micrornas with polysomes reflects distinct strengths of interactions with their mrna targets.</a> RNA. 18, 1612-1623 (2012).</li><li>Maroney, P. A., Yu, Y., Fisher, J., Nilsen, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+micrornas+are+associated+with+translating+messenger+rnas+in+human+cells.">Evidence that micrornas are associated with translating messenger rnas in human cells.</a> Nature Structural and Molecular Biology. 13, 1102-1107 (2006).</li><li>Paul, P., Chakraborty, A., Sarkar, D., Langthasa, M., Rahman, M., Bari, M., Singha, R. S., Malakar, A. K., Chakraborty, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+mirnas+and+human+diseases.">Interplay between mirnas and human diseases.</a> Journal of Cellular Physiology. 233, 2007-2018 (2018).</li><li>Rupaimoole, R., Slack, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microrna+therapeutics:+Towards+a+new+era+for+the+management+of+cancer+and+other+diseases.">Microrna therapeutics: Towards a new era for the management of cancer and other diseases.</a> Nature Reviews Drug Discovery. 16, 203-222 (2017).</li><li>Nelson, P. T., Hatzigeorgiou, A. G., Mourelatos, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mirnp:Mrna+association+in+polyribosomes+in+a+human+neuronal+cell+line.">Mirnp:Mrna association in polyribosomes in a human neuronal cell line.</a> RNA. 10, 387-394 (2004).</li><li>Nottrott, S., Simard, M. J., Richter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+let-7a+mirna+blocks+protein+production+on+actively+translating+polyribosomes.">Human let-7a mirna blocks protein production on actively translating polyribosomes.</a> Nature Structural and Molecular Biology. 13, 1108-1114 (2006).</li><li>Kim, J., Krichevsky, A., Grad, Y., Hayes, G. D., Kosik, K. S., Church, G. M., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+many+micrornas+that+copurify+with+polyribosomes+in+mammalian+neurons.">Identification of many micrornas that copurify with polyribosomes in mammalian neurons.</a> Proceedings of the National Academy of Sciences of the United States of America. , 360-365 (2004).</li><li>Androsavich, J. R., Chau, B. N., Bhat, B., Linsley, P. S., Walter, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disease-linked+microrna-21+exhibits+drastically+reduced+mrna+binding+and+silencing+activity+in+healthy+mouse+liver.">Disease-linked microrna-21 exhibits drastically reduced mrna binding and silencing activity in healthy mouse liver.</a> RNA. 18, 1510-1526 (2012).</li><li>Kraushar, M. L., Thompson, K., Wijeratne, H. R., Viljetic, B., Sakers, K., Marson, J. W., Kontoyiannis, D. L., Buyske, S., Hart, R. P., Rasin, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally+defined+neocortical+translation+and+polysome+assembly+are+determined+by+the+rna-binding+protein+hu+antigen+r.">Temporally defined neocortical translation and polysome assembly are determined by the rna-binding protein hu antigen r.</a> Proceedings of the National Academy of Sciences of the United States of America. , E3815-E3824 (2014).</li><li>McClatchy, D. B., Ma, Y., Liu, C., Stein, B. D., Martinez-Bartolome, S., Vasquez, D., Hellberg, K., Shaw, R. J., Yates, 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=Pulsed+azidohomoalanine+labeling+in+mammals+(palm)+detects+changes+in+liver-specific+lkb1+knockout+mice.">Pulsed azidohomoalanine labeling in mammals (palm) detects changes in liver-specific lkb1 knockout mice.</a> Journal of Proteome Research. 14, 4815-4822 (2015).</li><li>Schiapparelli, L. M., McClatchy, D. B., Liu, H. H., Sharma, P., Yates, J. R., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+detection+of+biotinylated+proteins+by+mass+spectrometry.">Direct detection of biotinylated proteins by mass spectrometry.</a> Journal of Proteome Research. 13, 3966-3978 (2014).</li><li>Wi&#347;niewski, J. R., Zougman, A., Nagaraj, N., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+sample+preparation+method+for+proteome+analysis.">Universal sample preparation method for proteome analysis.</a> Nature Methods. 6, 359-362 (2009).</li><li>Rajalingam, D., Loftis, C., Xu, J. J., Kumar, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trichloroacetic+acid-induced+protein+precipitation+involves+the+reversible+association+of+a+stable+partially+structured+intermediate.">Trichloroacetic acid-induced protein precipitation involves the reversible association of a stable partially structured intermediate.</a> Protein Science. 18, 980-993 (2009).</li><li>Fic, E., Kedracka-Krok, S., Jankowska, U., Pirog, A., Dziedzicka-Wasylewska, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+protein+precipitation+methods+for+various+rat+brain+structures+prior+to+proteomic+analysis.">Comparison of protein precipitation methods for various rat brain structures prior to proteomic analysis.</a> Electrophoresis. 31, 3573-3579 (2010).</li><li>Sashital, D. G., Greeman, C. A., Lyumkis, D., Potter, C. S., Carragher, B., 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=A+combined+quantitative+mass+spectrometry+and+electron+microscopy+analysis+of+ribosomal+30s+subunit+assembly+in+e.+Coli.">A combined quantitative mass spectrometry and electron microscopy analysis of ribosomal 30s subunit assembly in e. Coli.</a> Elife. 3, (2014).</li><li>Liang, S., Bellato, H. M., Lorent, J., Lupinacci, F. C. S., Oertlin, C., van Hoef, V., Andrade, V. P., Roff&#233;, M., Masvidal, L., Hajj, G. N. M., Larsson, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome-profiling+in+small+tissue+samples.">Polysome-profiling in small tissue samples.</a> Nucleic Acids Research. 46, e3 (2018).</li></ol>

Polysome perfil análisis permite el estudio de la traducción de proteínas mediante el análisis del estado traslacional de un mARN específico o el transcriptoma conjunto6,7. También es de gran ayuda cuando traducción local debe estudiarse como sinaptosomas8. Tradicionalmente, este método implica la separación de mono - y Polirribosomas y los mRNAs asociados a un gradiente de sacarosa que podría acoplarse con genómica o proteómic...

El corazón responde a la lesión de isquemia (I) y (R) la reperfusión de manera dinámica. Sin embargo, hay poca penetración en cambios agudos en la síntesis de proteínas durante la respuesta. Para abordar esto, aprovechamos el método bien establecido de polysome perfiles1 para identificar los cambios en la abundancia de proteínas que reflejan la redistribución de los ribosomas y factores reguladores traslacionales de cytosol a polisomas, y la aumento de las proteínas recién sintetizadas (NSPs). En el marco de I / R, el aumento en la síntesis de proteínas nuevas se produce en un marco de tiempo que es inconsistente con la transcripción de mRNAs nuevo2; Además, la discordancia entre los niveles de expresión de mRNA y proteína abundancia ha sido reportado3. Por estas razones, hemos optado por analizar los cambios en el proteoma dinámico como se refleja por la traducción de proteínas. Para ello, cuantificar el mRNA en las fracciones polysome y analizar la composición de la proteína en las fracciones polysome. Por último, porque microRNAs (miRs) regulan la disponibilidad de los mRNAs para la traducción y puede interferir con la eficacia de la proteína traducción4,5, examinamos la distribución de miRs en las fracciones polysome, centrándose en la respuesta a me / r.
Optamos por utilizar el modelo de perfusión en ratones aislados Langendorff y tejido cosechada bajo condiciones basales de perfusión continua, después de isquemia caudal global de 30 minutos y después de 30 min de isquemia seguida de 30 minutos de la reperfusión. A continuación solubilizado el tejido del corazón y separa polisomas sobre un gradiente de sacarosa, seguido del análisis proteómico y detección selectiva de mRNAs y miRNAs mediante PCR y microarrays, respectivamente. Esta combinación de métodos representa un enfoque poderoso para entender el proteoma dinámico, que permite la detección simultánea de MARN, miRNA y NSPs, así como la redistribución de las proteínas reguladoras, miRNA y mRNA entre fracciones de nontranslating, baja eficiencia polisomas y polisomas de alta eficiencia (ver figura 1). Penetraciones en la regulación dinámica de este proceso se extenderá por más análisis de la fosforilación de factores reguladores claves eIF2α como mTOR. Continuación se describen estos pasos individuales en detalle.

<table>
	<tbody>
		<tr>
			<td>Pentobarbital</td>
			<td>Vortech Phamaceuticals</td>
			<td>9373</td>
			<td>for euthansia</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sagent</td>
			<td>103424</td>
			<td>used in langendorff preparation</td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>Fine Science Tools</td>
			<td>91110-10</td>
			<td>used to hang the heart</td>
		</tr>
		<tr>
			<td>Langendorff system</td>
			<td>Radnoti + home made</td>
			<td>n/a</td>
			<td>A &#39;four heart&#39; system consisting of custom blown glass, tubing and water baths</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653-5KG</td>
			<td>Krebs buffer and Sucrose gradient</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td>Krebs buffer and Lysis buffer</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>P-5504</td>
			<td>Krebs buffer</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>M7774-500G</td>
			<td>Krebs buffer</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G5767</td>
			<td>Krebs buffer</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C1016-500G</td>
			<td>Krebs buffer</td>
		</tr>
		<tr>
			<td>Sucrose powder</td>
			<td>Sigma</td>
			<td>S0389-1KG</td>
			<td>Sucrose gradient</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>208337</td>
			<td>Sucrose gradient and Lysis buffer</td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td>Sigma</td>
			<td>T1503-1KG</td>
			<td>Sucrose gradient and Lysis buffer</td>
		</tr>
		<tr>
			<td>Xylene Cyanole</td>
			<td>Sigma</td>
			<td>X-4126</td>
			<td>Sucrose gradient</td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma-aldrich</td>
			<td>239763</td>
			<td>Sucrose gradient and Lysis buffer</td>
		</tr>
		<tr>
			<td>RNaseOUT</td>
			<td>Life Technologies</td>
			<td>C00019</td>
			<td>RNAse inhibitor for Lysis buffer</td>
		</tr>
		<tr>
			<td>Igepal CA-360 (NP40)</td>
			<td>Sigma</td>
			<td>I3021</td>
			<td>Lysis buffer</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail tablets, EDTA free</td>
			<td>Roche</td>
			<td>5056489001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube, Thinwall, Ultra-Clear, 13.2 mL, 14 x 89 mm</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman</td>
			<td>LE-80K</td>
			<td>Ultracentrifugation of the gradients</td>
		</tr>
		<tr>
			<td>Rotor</td>
			<td>Beckman</td>
			<td>SW41</td>
			<td>Ultracentrifugation of the gradients</td>
		</tr>
		<tr>
			<td>Biologic LP (pump)</td>
			<td>Biorad</td>
			<td>731-8300</td>
			<td>Fractionation of the gradients</td>
		</tr>
		<tr>
			<td>BioFrac</td>
			<td>Biorad</td>
			<td>741-0002</td>
			<td>Fractionation of the gradients</td>
		</tr>
		<tr>
			<td>Eppendorf RNA/DNA LoBind microcentrifuge tubes, 2 mL tube</td>
			<td>Sigma</td>
			<td>Z666513-100EA</td>
			<td>Gradient fraction and RNA extraction</td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Life technologies</td>
			<td>AM9738</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Luciferase Control RNA</td>
			<td>Promega</td>
			<td>L4561</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>C606-4</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Glycogen, RNA grade</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0551</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma</td>
			<td>I9516</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma</td>
			<td>E7023-1L</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>iScript cDNA Synthesis Kit</td>
			<td>BioRad</td>
			<td>170-8891</td>
			<td>Reverse transcription</td>
		</tr>
		<tr>
			<td>iTaq Universal SYBR Green Supermix</td>
			<td>BioRad</td>
			<td>175-5122</td>
			<td>Quantative PCR</td>
		</tr>
		<tr>
			<td>miRNeasy Micro Kit (50)</td>
			<td>Qiagen</td>
			<td>217084</td>
			<td>Kit for total RNA isolation</td>
		</tr>
		<tr>
			<td>miScript II RT Kit (50)</td>
			<td>Qiagen</td>
			<td>218161</td>
			<td>Kit for miRNA reverse transcription</td>
		</tr>
		<tr>
			<td>miScript Sybr Green PCR Kit (200)</td>
			<td>Qiagen</td>
			<td>218073</td>
			<td>Kit for real-time PCR expression analysis of miRNAs</td>
		</tr>
		<tr>
			<td>Centrifuge 5424R</td>
			<td>Eppendorf</td>
			<td></td>
			<td>For centrifugation of 1.5ml or 2.0ml tubes at different temperatures. Max speed - 21130g</td>
		</tr>
		<tr>
			<td>Centrifuge 5810R</td>
			<td>Eppendorf</td>
			<td></td>
			<td>For real-time PCR plate centrifugation at different temperatures. Max speed - 2039g</td>
		</tr>
		<tr>
			<td>My Cycler Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>For reverse transcription</td>
		</tr>
		<tr>
			<td>CFX96 Real-Time System/C1000 Touch Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>For real-time PCR analysis</td>
		</tr>
		<tr>
			<td>miRNeasy Serum/Plasma Spike-in Control</td>
			<td>Qiagen</td>
			<td>219610</td>
			<td>For quality control of RNA isolation</td>
		</tr>
		<tr>
			<td>Hard-Shell 96-Well PCR Plates, low profile, thin wall, skirted, green/clear</td>
			<td>Bio-Rad</td>
			<td>HSP9641</td>
			<td>For real-time PCR analysis</td>
		</tr>
		<tr>
			<td>Microseal &#39;B&#39; PCR Plate Sealing Film, adhesive, optical</td>
			<td>Bio-Rad</td>
			<td>MSB1001</td>
			<td>For real-time PCR plate sealing</td>
		</tr>
		<tr>
			<td>Research plus Single-Channel Pipette, Gray; 0.5-10 &#181;L</td>
			<td>Eppendorf</td>
			<td>UX-24505-02</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>PIPETMAN Classic Pipets, P20</td>
			<td>Gilson</td>
			<td>F123600G</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>PIPETMAN Classic Pipets, P200</td>
			<td>Gilson</td>
			<td>F144565</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>Rainin L-1000XLS Pipet-Lite XLS LTS Pipette 100-1000 &#181;L</td>
			<td>Gilson</td>
			<td>17011782</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>Glycogen, RNA grade</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0551</td>
			<td>Improves total RNA isolation efficiency</td>
		</tr>
		<tr>
			<td>Posi-Click 1.7 mL Tubes, natural color</td>
			<td>Denville</td>
			<td>C2170</td>
			<td>RNA isolation and storage; reagent mix</td>
		</tr>
		<tr>
			<td>Thermal Cycling Tubes -0.2 mL Individual Caps, Standard 0.2 mL tubes with optically</td>
			<td>Denville</td>
			<td>C18098-4 (1000910)</td>
			<td>Reverse transcription reaction</td>
		</tr>
		<tr>
			<td>Sharp 10 Precision barrier Tips</td>
			<td>Denville</td>
			<td>P1096-FR</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>Sharp 20 Precision barrier Tips</td>
			<td>Denville</td>
			<td>P1121</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>Sharp 200 Precision barrier Tips</td>
			<td>Denville</td>
			<td>P1122</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>Tips LTS 1 mL Filter</td>
			<td>Rainin</td>
			<td>RT-L1000F</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>miScript Primer Assay (200)</td>
			<td>Qiagen</td>
			<td>(it changes according to the miRNA)</td>
			<td>For real-time PCR analysis</td>
		</tr>
		<tr>
			<td>Gradient Master ver 5.3 Model 108</td>
			<td>BioComp Instruments</td>
			<td></td>
			<td>For preparation of sucrose gradients</td>
		</tr>
		<tr>
			<td>trichloroacetic acid</td>
			<td>Sigma Aldrich</td>
			<td>T6399</td>
			<td></td>
		</tr>
		<tr>
			<td>acetone</td>
			<td>Sigma Aldrich</td>
			<td>650501</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris hydrochloride</td>
			<td>Amresco</td>
			<td>M108</td>
			<td></td>
		</tr>
		<tr>
			<td>dithiothreitol</td>
			<td>Fisher Scientific</td>
			<td>BP172</td>
			<td></td>
		</tr>
		<tr>
			<td>iodoacetamide</td>
			<td>Gbiosciences</td>
			<td>RC-150</td>
			<td></td>
		</tr>
		<tr>
			<td>sequencing grade modified trypsine, porcine</td>
			<td>Promega</td>
			<td>V5111</td>
			<td></td>
		</tr>
		<tr>
			<td>ammonium bicarbonate</td>
			<td>BDH</td>
			<td>BDH9206</td>
			<td></td>
		</tr>
		<tr>
			<td>formic acid, Optima LC/MS</td>
			<td>Fisher Chemical</td>
			<td>A117</td>
			<td></td>
		</tr>
		<tr>
			<td>methanol, Optima LC/MS</td>
			<td>Fisher Chemical</td>
			<td>A454</td>
			<td></td>
		</tr>
		<tr>
			<td>acetonitrile, Optima LC/MS</td>
			<td>Fisher Chemical</td>
			<td>A996</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind tubes 0.5 mL</td>
			<td>Eppendorf AG</td>
			<td>22431064</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind tubes 1.5 mL</td>
			<td>Eppendorf AG</td>
			<td>22431081</td>
			<td></td>
		</tr>
		<tr>
			<td>HLB &#181;Elution plate 30 &#181;m</td>
			<td>Oasis</td>
			<td>186001828BA</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedVac concentrator</td>
			<td>Thermo Scientific</td>
			<td>Savant SPD2010</td>
			<td></td>
		</tr>
		<tr>
			<td>sonicator</td>
			<td>Qsonica</td>
			<td>Oasis180</td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>Thermo Scientific</td>
			<td colspan="2">Sorvall Legend micro 21R</td>
		</tr>
		<tr>
			<td>LC trap column PepMap 100 C18</td>
			<td>Thermo Scientific</td>
			<td>160454</td>
			<td></td>
		</tr>
		<tr>
			<td>LC separation column PepMap RSLC C18</td>
			<td>Thermo Scientific</td>
			<td>164536</td>
			<td></td>
		</tr>
		<tr>
			<td>mass spectrometer</td>
			<td>Thermo Scientific</td>
			<td colspan="2">Orbitrap Elite ion trap mass spectrometer</td>
		</tr>
		<tr>
			<td>MSConvert software</td>
			<td>ProteoWizard Toolkit</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sorcerer-SEQUEST software</td>
			<td>Sage-N Research, Inc.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

dynamic proteomic and mirna analysis of polysomes from isolated mouse heart after langendorff perfusion

Estudios en cambios dinámicos en la traducción de la proteína requieren métodos especializados. Aquí examinamos cambios en las proteínas recién sintetizadas en respuesta a la isquemia y reperfusión con el corazón de ratón perfundido aislado juntado con perfiles polysome. Para entender más los cambios dinámicos en la traducción de la proteína, caracteriza los mRNAs que fueron cargados con los ribosomas citosólicas (Polirribosomas o polisomas) mitocondriales polisomas se recuperó y en comparación con la distribución de mRNA y proteína en la fracciones de alta eficiencia (numerosos ribosomas ARNm), baja eficiencia (menos ribosomas) mitocondriales polisomas, y las fracciones no traducir. también puede asociar miRNAs mRNAs que se están traduciendo, reduciendo así la eficiencia de la traducción, examinamos la distribución de los miRNAs a través de las fracciones. La distribución de proteínas, mRNAs y miRNAs fue examinada bajo condiciones perfusión basals, al final de 30 minutos de isquemia global sin flujo y después de 30 minutos de la reperfusión. Aquí presentamos los métodos utilizados para llevar a cabo este análisis, en particular, el enfoque de optimización de la extracción de la proteína de la gradiente de sacarosa, ya que esto no se ha descrito antes y algunos resultados representativos.

Análisis del mRNA resultados de mRNA se pueden expresar como una distribución de un determinado mRNA en cada fracción (Figura 3A); para la cuantificación, combinar fracciones traducción polyribosomal y comparar a la fracción no traducir (figura 3B), presentando un cociente de abundancia del mRNA en la traducción de fracciones nontranslating. Información adicional es obtenida mediante el examen de las fraccione...

Watch this Scientific Journal Video about Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion at JoVE.com

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Aquí presentamos un protocolo para realizar polysome perfilado en el corazón de ratón perfundido aislado. Se describen métodos para la perfusión del corazón, polysome perfiles y análisis de las fracciones polysome mRNAs, miRNAs y el proteoma polysome.

Proteómicos y miRNA análisis de polisomas de corazón de ratón aislados después de Langendorff la perfusión

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

Research

JoVE Journal

Biochemistry

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Resolver purificado por afinidad Complejos de Proteína de Blue nativo PAGE y de proteínas de perfiles de correlación

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Investigación

Bioquímica

Dissection of Human Retina and RPE-Choroid for Proteomic Analysis

Disección de la Retina humana y RPE-coroides para análisis proteómico

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular ...

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

Purificación de hepatocitos y células endoteliales sinusoidales del hígado de ratón de perfusión

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

Split-BioID &#8212; Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

Split-BioID ÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã‚Â Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

Split-BioID: Análisis proteómico de complejos de proteínas específicos de contexto en su entorno celular nativo

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced ...

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

Análisis complejos de la proteína dinámica montan en y de interferometría de biocapa Biosensor usando espectrometría de masas y microscopia electrónica

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

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

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

Calibración-libre In Vitro cuantificación de proteína Homo-Oligomerización utilizando instrumentación comercial y Software de análisis de brillo libre de código abierto

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

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Análisis proteómico de polarización macrófago humano bajo un ambiente de poco oxígeno

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. ...

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Perfilado de Ubiquitina y Ubiquitina-como Modificaciones Post-traduccionales dependientes e Identificación de Alteraciones Significativas

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Proteomic Profile of EPS-Urine through FASP Digestion and Data-Independent Analysis

Perfil proteómico de EPS-Urine a través de la Digestión FASP y análisis independiente de datos

Filter-aided sample protocol (FASP) is widely used for proteomics sample preparation because it allows to concentrate diluted samples and it is compatible ...

F&#246;rster Resonance Energy Transfer Measurements in Living Plant Cells

FÃƒÆ’Ã‚Â¶rster Resonance Energy Transfer Measurements in Living Plant Cells

Mediciones de transferencia de energía por resonancia de Förster en células vegetales vivas

Sensitized emission-based F&#246;rster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser ...