Name: in vitro sumoylation assay to study sumo e3 ligase activity
Uploaded: 2018-01-29T12:30:00
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Este trabajo fue apoyado por subvenciones del Ministerio de ciencia y tecnología (la mayoría, 105-2320-B-010-007-MY3 para PCC), del Instituto Nacional de investigación de salud (INDH-EX105 10215BC para PCC), del Ministerio de ciencia y tecnología (más 105-2314-B-400-019 a HJK) y del Instituto de investigación nacional de salud (INDH MG-105-SP-10, INDH MG-106-SP-10 HJK). Este trabajo también fue financiado en parte con la Universidad Nacional de Yang Ming en la publicación del manuscrito al PCC. Los fundadores no tenían ningún papel en el diseño del estudio, recopilación de datos y análisis, publicación o reparación del manuscrito.

1. preparación de expresión de Baculovirus construye
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Purificar el SUMO E3 ligasa bZIP K mediante la clonación de cDNA de bZIP K19 en un vector del baculovirus de expresión dual (véase tabla de materiales) con un N-terminal del epítopo-tag. Hemos tenido éxito utilizando una etiqueta octapéptido (indicado en el protocolo como vector-etiqueta-K-bZIP). Nota: La plantilla de ADN para la clonación de la reacción en cadena (PCR) K-bZIP polimerasa es inversa del cD...

<ol>
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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=Role+of+SUMO-interacting+motif+in+Daxx+SUMO+modification,+subnuclear+localization,+and+repression+of+sumoylated+transcription+factors.">Role of SUMO-interacting motif in Daxx SUMO modification, subnuclear localization, and repression of sumoylated transcription factors.</a> Mol Cell. 24 (3), 341-354 (2006).</li><li>Welsh, M., Jamalpour, M., Zang, G., Akerblom, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+Src+Homology-2+domain+containing+protein+B+(SHB)+in+beta+cells.">The role of the Src Homology-2 domain containing protein B (SHB) in beta cells.</a> J Mol Endocrinol. 56 (1), 21-31 (2016).</li><li>Huang, W. Q., 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=Structure,+function,+and+pathogenesis+of+SHP2+in+developmental+disorders+and+tumorigenesis.">Structure, function, and pathogenesis of SHP2 in developmental disorders and tumorigenesis.</a> Curr Cancer Drug Targets. 14 (6), 567-588 (2014).</li><li>Perry, J. J., Tainer, J. A., Boddy, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+SIM-ultaneous+role+for+SUMO+and+ubiquitin.">A SIM-ultaneous role for SUMO and ubiquitin.</a> Trends Biochem Sci. 33 (5), 201-208 (2008).</li><li>Sun, H., Leverson, J. D., Hunter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+function+of+RNF4+family+proteins+in+eukaryotes:+targeting+a+ubiquitin+ligase+to+SUMOylated+proteins.">Conserved function of RNF4 family proteins in eukaryotes: targeting a ubiquitin ligase to SUMOylated proteins.</a> EMBO J. 26 (18), 4102-4112 (2007).</li><li>Ouyang, J., Shi, Y., Valin, A., Xuan, Y., Gill, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+binding+of+CoREST1+to+SUMO-2/3+contributes+to+gene-specific+repression+by+the+LSD1/CoREST1/HDAC+complex.">Direct binding of CoREST1 to SUMO-2/3 contributes to gene-specific repression by the LSD1/CoREST1/HDAC complex.</a> Mol Cell. 34 (2), 145-154 (2009).</li><li>Lyst, M. J., Stancheva, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+SUMO+modification+in+transcriptional+repression+and+activation.">A role for SUMO modification in transcriptional repression and activation.</a> Biochem Soc Trans. 35, 1389-1392 (2007).</li><li>Liu, H. W., 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=Chromatin+modification+by+SUMO-1+stimulates+the+promoters+of+translation+machinery+genes.">Chromatin modification by SUMO-1 stimulates the promoters of translation machinery genes.</a> Nucleic Acids Res. 40 (20), 10172-10186 (2012).</li><li>Rosonina, E., Duncan, S. M., Manley, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO+functions+in+constitutive+transcription+and+during+activation+of+inducible+genes+in+yeast.">SUMO functions in constitutive transcription and during activation of inducible genes in yeast.</a> Genes Dev. 24 (12), 1242-1252 (2010).</li><li>Chang, P. 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=The+chromatin+modification+by+SUMO-2/3+but+not+SUMO-1+prevents+the+epigenetic+activation+of+key+immune-related+genes+during+Kaposi's+sarcoma+associated+herpesvirus+reactivation.">The chromatin modification by SUMO-2/3 but not SUMO-1 prevents the epigenetic activation of key immune-related genes during Kaposi's sarcoma associated herpesvirus reactivation.</a> BMC Genomics. 14, 824 (2013).</li><li>Neyret-Kahn, H., 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=Sumoylation+at+chromatin+governs+coordinated+repression+of+a+transcriptional+program+essential+for+cell+growth+and+proliferation.">Sumoylation at chromatin governs coordinated repression of a transcriptional program essential for cell growth and proliferation.</a> Genome Res. 23 (10), 1563-1579 (2013).</li><li>Tatham, M. H., 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=Polymeric+chains+of+SUMO-2+and+SUMO-3+are+conjugated+to+protein+substrates+by+SAE1/SAE2+and+Ubc9.">Polymeric chains of SUMO-2 and SUMO-3 are conjugated to protein substrates by SAE1/SAE2 and Ubc9.</a> J Biol Chem. 276 (38), 35368-35374 (2001).</li><li>Mattoscio, D., Segre, C. V., Chiocca, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+manipulation+of+cellular+protein+conjugation+pathways:+The+SUMO+lesson.">Viral manipulation of cellular protein conjugation pathways: The SUMO lesson.</a> World J Virol. 2 (2), 79-90 (2013).</li><li>Gareau, J. R., Lima, C. 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El en vitro la sumoilación protocolo descrito aquí se utiliza rutinariamente para establecer la situación de la sumoilación de sustratos Ubc9 identificados. La limitación principal utilizando el protocolo estándar para el estudio de la función de SUMO E3 ligasa de sumoilación es la abundancia de activación de SUMO E1 y E2 Conjugación enzimas Ubc9 que maximicen la conjugación de SUMO en sistemas in vitro . Teniendo en cuenta este reto, creemos la titulación de la cantidad de enzimas SUMO E1 y...

Modificación de SUMO fue identificado inicialmente como una modificación poste-de translación reversible (PTM) que regula la estabilidad de la proteína1. Además de conjugación directa, SUMO puede también acoplarse a una proteína a través de la interacción no covalente por SUMO interacción motivos (SIMs)2. Similar a la Unión de Tirosil-fosforilación de moléculas que de homología Src 2 (SH2) o phosphotyrosine atar (PTB) dominios3,4, modificación SUMO proporciona una plataforma de interacción adicional para selectivo contratación de SIM que contiene proteínas de efector5,6. Además de regulación de la transducción de la señal, la sumoilación de factores transcripcionales y cromatina remodelación moléculas sirven para modular la gene expresión7,8. Estudios de patrones de sumoilación de genoma han revelado que la modificación SUMO es asociada a positivo9,10 o negativo10,11,12 transcripción Reglamento, en parte debido a la especificidad del paralogue de sumoilación.
Hay tres isoformas principales de SUMO para la proteína conjugando presente en células de mamíferos; Estos incluyen SUMO-1 y la altamente homólogo SUMO-2 y SUMO-3 (denominado SUMO-2/3)13. SUMO se conjuga generalmente a los residuos de lisina (K) en el motivo de consenso ψKxD/E en la proteína diana. La SIM en SUMO E3 ligasas es responsable de la especificidad de paralogue14. En contraste con vías de ubiquitinación que contiene cientos de ligasas E3, sólo ha habido unos SUMO E3 ligasas identificados15. Ligasas de SUMO E3 se definen por propiedades, incluyendo su capacidad para (i) atar Ubc9, (ii) parte de SUMO se unen a través de un dominio SIM y (iii) mejorar la transferencia SUMO de Ubc9 sobre substrato. Ligasa E3 no es absolutamente necesario para SUMO verbal16, pero proporciona sustrato y SUMO paralogue especificidad. Puesto que generalmente sólo una pequeña fracción de la proteína total del sustrato es modificado de SUMO, detección de proteínas sumoiladas en vivo es siempre un reto. Por lo tanto, se necesitan análisis precisos y reproducibles para aclarar la función precisa de modificación de SUMO.
El en vitro la sumoilación ensayo, que evalúa la capacidad de Ubc9 purificada para catalizar la sumoilación de proteínas sustrato, es un ensayo bien-aceptado para el estudio de modificación SUMO17. Sin embargo, SUMO E3 ligasa actividad en la mayoría de los casos no puede ser detectada o solamente se pueden detectar con ligasa SUMO transformada con alta SUMO E3 ligasa actividad y especificidad de sustrato bajo el protocolo estándar debido a la presencia de una abundante cantidad de Ubc918 . El éxito de este ensayo depende en gran medida la valoración cuidadosa de Ubc9 purificada. El ensayo de la sumoilación en vitro descrito aquí se modifica una sumoilación estándar protocolo (véase Tabla de materiales). La observación de mayor modificación SUMO global durante la reactivación del herpesvirus asociado a sarcoma de Kaposi (KSHV) nos llevó a identificar la ligasa E3 SUMO viral que puede ser responsable de la para arriba-regulación de la sumoilación. Tras una proyección en pequeña escala de las proteínas obran recíprocamente de viral SUMO E3 ligasa K bZIP, p53 fue identificado como un sustrato nuevo. En este protocolo, se muestra detalladamente en células de los insectos los pasos involucrados en la expresión y purificación de tipo K-bZIP, una ligasa E3 SUMO viral con especificidad de SUMO-2/3. Se evalúa la capacidad de la K-bZIP purificado para mejorar la sumoilación de p53, un conocido sustrato SUMO, en presencia de cantidades reducidas de enzimas E1 y E2. Usando este análisis modificado de sumoilación, los investigadores pueden confiablemente definir las capacidades del SUMO E3 ligasa SUMOylate novela o sustratos conocidos, que es un paso primordial en el estudio de la sumoilación de la proteína. Además, este método ayuda a identificar nuevos ligasas SUMO E3 con actividad ligasa baja pero la especificidad de sustrato alta.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">pFastBac</td>
			<td style="width:153px;">Invitrogen</td>
			<td style="width:119px;">10359-016</td>
			<td style="width:219px;">dual expression baculovirus vector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pCR2.1-TOPO vector</td>
			<td style="width:153px;">Invitrogen</td>
			<td style="width:119px;"></td>
			<td>PCR product vector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">competent cell&#160; E. coli DH5&#945;</td>
			<td style="width:153px;">Yeastern Biotech</td>
			<td style="width:119px;">FYE678-80VL</td>
			<td>competent E. coli cells A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">E. coli DH10Bac</td>
			<td style="width:153px;">Invitrogen</td>
			<td style="width:119px;">10359-016</td>
			<td>competent E. coli cells B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">FugeneHD</td>
			<td style="width:153px;">Roche</td>
			<td style="width:119px;">04709705001</td>
			<td>transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Opti-MEM</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">31985062</td>
			<td>reduced serum media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">T4 Ligase</td>
			<td style="width:153px;">NEW England BioLabs</td>
			<td style="width:119px;">M0202S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">CpoI (RsrII)</td>
			<td style="width:153px;">Thermo Scientific</td>
			<td style="width:119px;">ER0741</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Bluo-gal</td>
			<td style="width:153px;">Thermo Scientific</td>
			<td style="width:119px;">B1690</td>
			<td>galactosidase substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">IPTG</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">I6758-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Grace&#8217;s Insect Medium</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">11605094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Fetal Bovine Serum</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">10082147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Gentamicin</td>
			<td style="width:153px;">Thermo Fisher</td>
			<td style="width:119px;">15750060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Ampicillin</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9393-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Kanamycin</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">K0254-20ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">gentamicin</td>
			<td style="width:153px;">Gibco</td>
			<td style="width:119px;">15710-064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">tetracyclin</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">87128-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">LB Broth</td>
			<td style="width:153px;">Merk</td>
			<td style="width:119px;">1.10285.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">HEPES</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">H4034-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">NaCl</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9888-5KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">KCl</td>
			<td style="width:153px;">Merk</td>
			<td style="width:119px;">1.04936.1000</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:363px;">Na2HPO4</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5136-500G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:363px;">KH2PO4</td>
			<td style="width:153px;">J.T.Baker</td>
			<td style="width:119px;">3246-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">sodium dodecyl sulfate</td>
			<td style="width:153px;">Merk</td>
			<td style="width:119px;">1.13760.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">&#946;-mercaptoethanol</td>
			<td style="width:153px;">Bio-Rad</td>
			<td style="width:119px;">161-0710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">TRIS (Base)</td>
			<td style="width:153px;">J.T.Baker</td>
			<td style="width:119px;">4109-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Non-fat milk</td>
			<td style="width:153px;">Fonterra</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">glycerol</td>
			<td style="width:153px;">J.T.Baker</td>
			<td style="width:119px;">2136-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Triton X-100</td>
			<td style="width:153px;">Amresco</td>
			<td style="width:119px;">0694-1L</td>
			<td>detergent A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Tween 20</td>
			<td style="width:153px;">Amresco</td>
			<td style="width:119px;">0777-500ML</td>
			<td>detergent B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Poloxamer 188 solution</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">P5556-100ML</td>
			<td>detergent C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Protease Inhibitor Cocktail Tablet</td>
			<td style="width:153px;">Roche</td>
			<td style="width:119px;">04 693 132 001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">3x Flag peptide</td>
			<td style="width:153px;">Sigma</td>
			<td style="width:119px;">F4799</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">anti-FLAG m2 Magnetic beads</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8823</td>
			<td>antibody-tagged magnetic beads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">SUMOlink SUMO-1 Kit</td>
			<td style="width:153px;">Active Motif</td>
			<td style="width:119px;">40120</td>
			<td>standard SUMOylation protocol&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">SUMOlink SUMO-2/3 Kit</td>
			<td style="width:153px;">Active Motif</td>
			<td style="width:119px;">40220</td>
			<td>standard SUMOylation protocol&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">QIAquick Gel Extraction Kit</td>
			<td style="width:153px;">QIAGEN</td>
			<td style="width:119px;">28704</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">QIAGEN Plasmid Mini Kit</td>
			<td style="width:153px;">QIAGEN</td>
			<td style="width:119px;">12123</td>
			<td>plasmid extraction kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Polypropylene tubes</td>
			<td style="width:153px;">Falcon</td>
			<td style="width:119px;">352059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Petri Dish</td>
			<td style="width:153px;">Falcon</td>
			<td style="width:119px;">351029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Cell lifter</td>
			<td style="width:153px;">Corning</td>
			<td style="width:119px;">CNG3008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Loading tip</td>
			<td style="width:153px;">Sorenson BioScience</td>
			<td style="width:119px;">28480</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">PVDF</td>
			<td style="width:153px;">PerkinElmer</td>
			<td style="width:119px;">NEF1002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Blotting filter paper</td>
			<td style="width:153px;">Bio-Rad</td>
			<td style="width:119px;">1703932</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Mini slab gel apparatus (Bio-Rad Mini Protean II Cell)</td>
			<td style="width:153px;">Bio-Rad</td>
			<td style="width:119px;">1658001 EDU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell</td>
			<td style="width:153px;">Bio-Rad</td>
			<td style="width:119px;">1703940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Pierce ECL Western Blotting</td>
			<td style="width:153px;">Thermo</td>
			<td style="width:119px;">32106</td>
			<td>ECL reagent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:363px;">suspension mixer</td>
			<td style="width:153px;">Digisystem laboratory instruments&#160; Inc.</td>
			<td style="width:119px;">SM-3000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">orbital shaker</td>
			<td>Kansin instruments Co.</td>
			<td>OS701</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:363px;">ImageQuant LAS 4000 
			biomolecular imager</td>
			<td style="width:153px;">GE Healthcare</td>
			<td style="width:119px;">28955810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Sf9</td>
			<td style="width:153px;">Thermo Scientific</td>
			<td>B82501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">anti-p53 antibody</td>
			<td>Cell Signaling</td>
			<td>#9282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">anti-rabbit antibody</td>
			<td>GE Healthcare</td>
			<td>NA934-1ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro sumoylation assay to study sumo e3 ligase activity

Modificación de pequeños ubiquitin-like modifier (SUMO) es una importante modificación poste-de translación (PTM) que media el transduction de la señal sobre todo a través de la modulación de las interacciones proteína-proteína. Similar a la modificación de la ubiquitina, la sumoilación está dirigida por una cascada secuencial de enzimas como enzima activadora de E1 (SAE1/SAE2), enzima E2-Conjugación (Ubc9) y E3 ligasa (es decir, Pia familia, RanBP2 y Pc2). Sin embargo, a diferencia de la ubiquitinación, una ligasa E3 es no esencial para la reacción pero no proporcionan precisión y eficacia para verbal de SUMO. Las proteínas modificadas por sumoilación pueden identificarse por ensayo en vivo mediante inmunoprecipitación con anticuerpos específicos de sustrato y el immunoblotting con anticuerpos específicos para SUMO. Sin embargo, la demostración de la actividad de la proteína SUMO E3 ligasa requiere reconstitución en vitro de sumoilación ensayos con enzimas purificadas, sustrato y proteínas SUMO. Ya que en las reacciones en vitro , generalmente SAE1/SAE2 y Ubc9, solo son suficientes para Conjugación de SUMO, realce de la sumoilación por una supuesta ligasa E3 no siempre es fácil de detectar. Aquí, describimos una modificada en vitro la sumoilación protocolo que identifica sistemáticamente modificación SUMO usando un sistema en vitro reconstituido. También se presenta un protocolo paso a paso para purificar catalítico activo K-bZIP, una ligasa E3 de SUMO-2/3 viral. Las actividades de la sumoilación de la purificada bZIP de K aparecen en p53, un conocido destino de SUMO. Este protocolo puede no sólo utilizarse para dilucidar las ligasas SUMO E3 novela, sino también para revelar su especificidad paralog SUMO.

Según la información proporcionada por el fabricante, la cantidad de enzima E1 y E2 en el ensayo de sumoilación es de 50 nM y 500 nM, respectivamente. Primero se determinó la cantidad mínima de E2 conjugando enzimas Ubc9 que es capaz de SUMOylate p53 por un análisis de la sumoilación en vitro . Tan bajo como un quinto de la cantidad de Ubc9 utilizado en el estándar en vitro la sumoilación protocolo eficiente SUMOylate p53 (figura 1<...

Watch this Scientific Journal Video about In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity at JoVE.com

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

A diferencia de ligasas de ubiquitina ligasas E3 SUMO algunos han sido identificados. Este modificado en vitro protocolo de sumoilación es capaz de identificar nuevos SUMO E3 ligasas por un ensayo de reconstitución en vitro .

In Vitro Ensayo de la sumoilación estudiar SUMO E3 ligasa actividad

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily ...

The overall goal of this experiment is to verify a protein of interest that can be SUMOylated or is a SUMO E3 lygase. This method can help answer key questions in the SUMOylation field, such as SUMO E3 lygase identification. The main advantage of this technique is that it could be utilized for investigation of SUMO E3 lygase specificity toward different SUMO iso forms.Demonstrating the procedure will be Wan-Shan Young, a PhD student from my laboratory. To purify the tag K-bZIP, first incubate 10 milliliters of cellular lysate with 50 microliters of antibody-tagged magnetic beads in a 14-milliliter polypropylene tube. Then rotate the tube at 50 revolutions per minute at four degrees Celsius for three hours in a suspension mixer.Once the protein is captured centrifuge the sample at 800 times g for 30 seconds at four degrees Celsius to pellet the beads. Discard all but one milliliter of supernatant to re-suspend the beads. And transfer the dissolved beads into a 1.5-milliliter tube.To wash the captured proteins, place the tube with the beads on a magnetic stand. Wait for about three seconds for the beads to adhere to the side walls of the tube and then remove the supernatant. To wash the beads, add one milliliter of lysis buffer into the 1.5-milliliter tube.Then invert the tube ten times. Next, the place the 1.5-milliliter tube on the magnetic stand and remove the supernatant once the beads adhere to the walls. To wash the beads again, add one milliliter of phosphate buffered saline to the 1.5 milliliter tube, and invert it ten times.Then transfer the tube to the magnetic stand, and remove the supernatant once the beads have attached to the side walls. To elute the K-bZIP tagged proteins from the antibody-tagged magnetic beads, add 100 microliters at 150 micrograms per milliliter of octapeptide diluted in phosphate buffered saline in the 1.5-milliliter tube. Then rotate the tube on a suspension mixer at 50 revolutions per minute for ten minutes at room temperature.Transfer the tube to the magnetic stand. And then collect the K-bZIP containing supernatant. Next, to analyze the purified K-bZIP tag, subject one to five microliters of the purified protein to SDS page, followed by Coomassie blue staining.Load 0.5, 1 and 2 micrograms of bovine serum albumin as standards on the gel for quantifying the K-bZIP concentration. While processing the image on Image J.Next, dilute the purified K-bZIP in phosphate buffered saline. To attain a final concentration of 100 nanograms per microliter.And store at minus 80 degrees Celsius in small aliquots. Add three microliters of 100 nanograms per microliter of purified tagged K-bZIP in the in vitro SUMOylation master mix reaction. Then mix the contents of the master mix gently and incubate at 30 degrees Celsius for three hours.Next, add 20 microliters of 2X SDS page with loading buffer to stop the reaction. Then heat the samples at 95 degrees Celsius for five minutes to denature the protein. Next, load 20 microliters of the sample on a 10%SDS page gel.And run the gel at 80 volts for about 120 minutes till the dye reaches the bottom of the gel. Once the electro paresis is over, disconnect the gel apparatus. And transfer the gel in semi-dry transfer buffer for five minutes.Then soak the PVDF membrane in another container filled with methanol for one minute. Remove the PVDF membrane from the methanol and soak in semi-dry transfer buffer. Then gently agitate the membrane for five minutes.Remove the safety cover of the semi-dry electrophoretic apparatus. Pre-wet a filter paper. And prepare a gel sandwich on the bottom of the platinum anode.After securing the cathode plate in the safety cover, run the blot at 15 volts constant current for 90 minutes. Then turn the power supply off and take the PVDF membrane out after disconnecting the semi-dry apparatus. Now block the PVDF membrane with blocking buffer for one hour at room temperature on an orbital shaker at 30 revolutions per minute.Next, hybridize the PVDF membrane with anti-p53 antibody and blocking buffer for 12 to 16 hours at four degrees Celsius on a suspension mixer at 30 revolutions per minute. Then take the PVDF membrane out and transfer in a container filled with Tris-buffered saline tween 20. Next, soak the PVDF membrane in Tris-buffered saline tween 20 for 30 minutes on the orbital shaker at 45 revolutions per minute.Then hybridize the PVDF membrane with anti-rabbit antibody conjugated with horseradish peroxidase diluted in blocking buffer for one hour at room temperature on the suspension mixer at 30 revolutions per minute. Next, wash the PVDF membrane with Tris-buffered saline tween 20 thrice. After washing, soak the PVDF membrane in Tris-buffered saline tween 20 for 30 minutes on the suspension mixer at 45 revolutions per minute.Then replace the Tris-buffered saline tween 20 with phosphate-buffered saline. To preserve the PVDF membrane at four degrees Celsius for up to 12 hours. Next, mix the enhanced chemiluminescent substrate reagents one and two in a one-to-one ratio.Then remove the PVDF membrane from the phosphate-buffered saline and blot briefly with the punch pockets to absorb the excess moisture. Immediately add 400 microliters of the Chemiluminescent reagent to the membrane surface and wait for three to five minutes. Briefly blot to drain the excess Chemiluminescent reagent, preventing the membrane from drying completely.Use Luminescent's imaging system to expose the blot. Here immuno blot analysis was done to show the concentration of Ubc9 enzyme required to SUMOylate the p53 targets. This shows that 1/2 and 1/5 the concentration of Ubc9 enzyme can sufficiently SUMOylate p53 enzyme in an in vitro SUMOylation assay using SUMO 1 and SUMO 2 peptides.To analyze the SUMOylated p53 the membrane was probed with anti-p53 antibody. Similar immuno blot analysis was done to show that half of E1 and 1/10 concentration of Ubc9 enzymes are required to SUMOylate the p53 target in an in vitro SUMOylation assay using SUMO 1, SUMO 2 and SUMO 3 peptides. Here the membrane was stained with Coomassie to show the purified K-bZIP protein.Baculovirus expression system was used to express the purified K-bZIP which was analyzed on SDS page with bovine serum albumin as standard for comparison. Additional immuno blot analysis was also done which showed that on increasing the concentration of K-bZIP it catalyzes the SUMOylation of p53 in the presence of half the amount of E1, and 1/10 the amount of Ubc9 enzymes, respectively. Once mastered, this technique can be done in four hours if it is performed properly.While attempting this procedure it is important to remember to keep all the reagent for the in vitro SUMOylation assay on ice, and to do the series dilution rather than in one giant dilution. Find this procedure other methods like over expression SUMOlygase can be performed in order to verify the SUMOylation in vitro. After its development, this technique paved the way for researchers in the field of SUMOylation to explore more SUMOlygase and validate their power of specificity.After watching this video, you should have a good understanding of how to identify a SUMO e3 lygase using in vitro simulation assay.

in-vitro-sumoylation-assay-to-study-sumo-e3-ligase-activity

Research

JoVE Journal

Biology

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Prueba de sensibilidad visual a la velocidad y dirección del movimiento en lagartos

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Investigación

Biología

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

Un In vitro Tumor FluoroBlok invasión de ensayo

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

Ensayo de la actividad quinasa de LRRK2 In vitro</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Ensayo de formación de tubos de células endoteliales para la<em&gt; In Vitro</em&gt; Estudio de la angiogénesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

In vitro metilación de ensayo para estudiar la proteína arginina metilación

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.

Ensayo in vitro para medir Fosfatidiletanolamina metiltransferasa actividad

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from ...

Measuring In Vitro ATPase Activity for Enzymatic Characterization

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization. Proteins hydrolyze ATP in a reaction that results in inorganic phosphate release, and the amount of phosphate liberated is then quantitated using a colorimetric assay. This highly adaptable protocol can be adjusted to measure ATPase activity in kinetic or endpoint assays. A representative protocol is provided here based on the activity and requirements of EpsE, the AAA+ ATPase involved in Type II Secretion in the bacterium Vibrio cholerae. The amount of purified protein needed to measure activity, length of the assay and the timing and number of sampling intervals, buffer and salt composition, temperature, co-factors, stimulants (if any), etc. may vary from those described here, and thus some optimization may be necessary. This protocol provides a basic framework for characterizing ATPases and can be performed quickly and easily adjusted as necessary.

Measuring In Vitro ATPase Activity for Enzymatic Characterization

We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.

Medición<em&gt; In Vitro</em&gt; Actividad ATPasa para enzimática Caracterización

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate ...

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, small ubiquitin-related modifier (SUMO) is a ubiquitin-like protein that is covalently attached to the lysine residues of a variety of target proteins to regulate cellular processes, such as gene transcription, DNA repair, protein interaction and degradation, subcellular transport, and signal transduction. The most common approach to detecting protein SUMOylation is based on the expression and purification of recombinant tagged proteins in bacteria, allowing for an in vitro biochemical reaction which is simple and suitable for addressing mechanistic questions. However, due to the complexity of the process of SUMOylation in vivo, it is more challenging to detect and analyze protein SUMOylation in cells, especially when under endogenous conditions. A recent study by the authors of this paper revealed that endogenous retinoblastoma (Rb) protein, a tumor suppressor that is vital to the negative regulation of the cell cycle progression, is specifically SUMOylated at the early G1 phase. This paper describes a protocol for the detection and analysis of Rb SUMOylation under both endogenous and exogenous conditions in human cells. This protocol is appropriate for the phenotypical and functional investigation of the SUMO-modification of Rb, as well as many other SUMO-targeted proteins, in human cells.

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Small ubiquitin-related modifier (SUMO) family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. This paper describes a protocol for the detection of retinoblastoma (Rb) protein SUMOylation under endogenous and exogenous conditions in human cells.

En Vivo Detección y análisis de la sumoilación de la proteína Rb en células humanas

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, ...

Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the prediction of family functions based on several features, such as the presence of sequence motifs or of particular sites for post-translational modification and the expression profile of family members in different conditions. This work describes a detailed protocol for gene family characterization. Here, the procedure is applied to the characterization of the Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligase family in grapevine. The methods include the genome-wide identification of family members, the characterization of gene localization, structure, and duplication, the analysis of conserved protein motifs, the prediction of protein localization and phosphorylation sites as well as gene expression profiling across the family in different datasets. Such procedure, which could be extended to further analyses depending on experimental purposes, could be applied to any gene family in any plant species for which genomic data are available, and it provides valuable information to identify interesting candidates for functional studies, giving insights into the molecular mechanisms of plant adaptation to their environment.

This article describes the procedure for the identification and characterization of a gene family in grapevine applied to the family of Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligases.

Flujo de trabajo integral para la identificación de todo el genoma y Meta-análisis de la expresión de la familia del Gene del ATL E3 ubiquitina ligasa en Grapevine

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the ...

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. These transport mechanisms are mediated by the activities of specialized transporter proteins. Specifically,pH dynamics are finely controlled by plasma membrane proton (H+) extrusion systems, such as the Na+/H+ exchanger (NHE) protein family. Despite extensive efforts to study the mechanisms underlying NHE regulation, our current understanding of the biophysical and molecular properties of the NHE family is inadequate because of the limited availability of methods to effectively measure NHE activity. In this manuscript, we used H+-selective electrodes during whole-cell patch clamping recording to measure NHE-induced H+ flux. We proposed this approach to overcome some limitations of typically used methods to measure NHE activity, such as radioactive uptake and fluorescent membrane permeants. Measurement of NHE activity using the described method enables high sensitivity and time resolution and more efficient control of intracellular H+ concentrations. H+-selective electrodes are based on the fact that transporter activity creates an ion gradient in close proximity to the cell membrane. An H+-selective electrode moving up to and away from the cell membrane in a repetitive, oscillatory fashion records a voltage difference that is dependent on H+ flux. While H+-selective electrodes are used to detect H+ flux moving out of the cell, the patch clamp method in the whole-cell configuration is used to control the intracellular ion composition. Moreover, application of the giant patch clamp technique allows modification of the intracellular composition of not only ions but also lipids. The transporter activity of NHE isoform 3 (NHE3) was measured using this technical approach to study the molecular basis of NHE3 regulation by phosphoinositides.

This manuscript describes the applications of proton-selective electrodes and patch clamping methods to measure the activity of proton transport systems. These methods overcome some limitations of techniques commonly used to study proton transport activity, such as moderate sensitivity, time resolution and insufficient intracellular milieu control.

Aplicación de medición de electrofisiología para estudiar la actividad de transportadores de Electro-neutro

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. ...