Este trabajo fue apoyado por becas de los Institutos Nacionales de Salud de VF (NS42599 y GM077569).

1. Preparación para la reticulación <ol><li> Será necesario que la placa de un número suficiente de células para permitir el aislamiento de 500 g de proteína por cada tubo de reacción estándar. Un único tubo puede ser suficiente para la identificación de los interactores putativo de una proteína de interés por inmunoblot. En este caso, material de cordón atado puede ser eluida con tampón de muestra SDS-PAGE (inmunoprecipitación). Para el análisis de espectrometría de masas, el número de reacciones estándar debe ser aumentado por lo menos diez veces y complejos de proteínas debe ser eluida por fuera de la competencia con un péptido antígeno reconocida por el anticuerpo utilizado (cromatografía de inmunoafinidad). Esta estrategia permitirá el aislamiento de los complejos de proteínas libres de inmunoglobulina. </li><li> La densidad óptima de la célula para la reticulación es entre 75% y el 90% de confluencia. En condiciones de alta confluencia, las células tienden a apilarse unos sobre otros, lo que disminuye la accesibilidad a la reticulante penetran en las células de todas las células. </li><li> Tipo de células y condiciones experimentales deben estar etiquetados en la parte inferior de la placa de la célula. En forma rutinaria se incluyen controles con el vehículo solo. </li><li> Prepare todas las soluciones, excepto la solución DSP antes de la reticulación. <ol><li> Prepare un inventario de tampón fosfato buffer salino con CaCl2 0,1 mM y 1 mM CaCl2 (PBS / Ca / Mg) antes de comenzar el experimento. La adición de iones calcio y magnesio es esencial para la adhesión de las células de la placa de cultivo durante el transcurso del experimento. Almacenar a 4 ° C. </li><li> 50X de la proteasa completo cóctel de inhibidores de - 1 tableta disuelta en 1 ml de agua Milli-Q. Almacenar a -20 ° C. </li><li> 20% de Triton X-100 Pesar 10g Triton X -100 y se diluye en el volumen total de 50 ml de agua Milli-Q. Rock en 4 ° C durante la noche y se almacenan a 4 ° C hasta su uso. No almacenar por más de 1 mes. Use las diluciones de esta población un 20% para preparar los tampones de lisis y IP se describen a continuación. </li><li> 50X DSP enfriamiento solución 1M Tris a pH 7,4. Conservar a temperatura ambiente. </li><li> Preparar un buffer 10x una solución. Buffer 10X A: <ul><li> 50 ml de 1 M HEPES </li><li> 150 ml de NaCl 5 M </li><li> 10 ml de 0,5 M EGTA </li><li> 250 l 2 M de MgCl2 </li><li> Ajustar el pH a 7.4 </li><li> Alcanzar un volumen final de 500 ml </li></ul> El Buffer 10X Una solución se diluye a 1X tampón A cuando sea necesario. Buffer 1x A también se usa en el lisado y tampones inmuno-magnética-precipitación. Un tampón 1X <ul><li> Hepes 10 mM </li><li> 150 mM NaCl </li><li> 1 mM EGTA </li><li> 0,1 mM de MgCl 2 </li><li> pH 7,4 </li></ul></li><li> Tampón de lisis 1X tampón A + 0,5% Triton X-100. </li><li> Inmuno-magnética tampón de (Buffer IP) 1X tampón A + 0,1% Triton X-100. </li></ol></li></ol> 2. Preparación de la solución de reticulación <ol><li> Prepare la solución DSP inmediatamente antes de aplicar a las células. DSP es altamente hidrofóbico y se disuelven en DMSO antes de diluir en PBS / Ca / Mg de amortiguamiento. Disolver 40 mg de DSP en 1 ml de DMSO. Esto hace que una solución 100 mM de DSP (El peso molecular del DSP es 404,42 g / mol). </li><li> Caliente un volumen adecuado de PBS / Ca / Mg a 37 ° C con el fin de facilitar la dilución de la DSP / DMSO en PBS. Volúmenes necesarios para una variedad de tamaños de placas: 6-así placa de 2 ml por pozo 10 cm placa de 10 ml por placa 15 cm placa de 20 ml por placa </li><li> Añadir 10μL de solución madre DSP / DMSO por cada 1 ml de PBS caliente / Ca / Mg. Añadir DSP / DMSO gota a gota solución de reserva con la mezcla repite hasta que todos los DSP se ha disuelto. Prepare una solución de control de 10 l DMSO añadido a cada 1 ml de PBS / Ca / Mg. </li><li> Lugar todas las soluciones de PBS / Ca / Mg en un baño de agua helada no más de 10 min. </li></ol> 3. Preparar las células para la reticulación <ol><li> Prepare un baño de agua helada que se ajuste a todas las placas para la reticulación o la incubación control del vehículo. </li><li> Tomar placas de la incubadora a 37 ° C y colocarlos inmediatamente en el baño de agua helada. </li><li> Lavar las células dos veces con helado de PBS / Ca / Mg. Usar el mismo volumen que va a utilizar para la incubación reticulante (ver sección 2.2). </li><li> Quitar segundo lavado y añadir ya sea el control del vehículo o la solución de DSP entrecruzamiento de amortiguamiento. </li><li> Incubar en hielo durante dos horas. Usted debe verificar las placas aproximadamente cada veinte minutos para asegurarse de que todas las células están cubiertos por la solución. Usted puede notar una pequeña cantidad de DSP se precipitan fuera de la solución. Esto es normal. </li></ol> 4. La inactivación de reacción DSP <ol><li> Prepare una solución 1X DSP de enfriamiento de 20 mM Tris pH 7,4 en PBS / Ca / Mg (20 l de 1 M Tris pH 7,4 por cada 1 ml de PBS / Ca / Mg). </li><li> Quitar el control del vehículo y las soluciones de reticulación. </li><li> Añadir helada solución inactivación e incubar en hielo durante 15 minutos. </li></ol> 5. Lisis celular <ol><li> Durante la incubación de enfriamiento DSP, preparar la solución amortiguadora de lisis celular de 0,5% Triton X-100 en tampón A con un cóctel completo inhibidor de la proteasa. Añadir 20 l solución de archivo de 50X completa para cada solución amortiguadora de lisis 1 mL. </li><li> Después de la incubación de 15 minutos de enfriamiento DSP, lavar las células dos veces con PBS / Ca / Mg. </li><li> Añadir tampón de lisis completa + a las células y el rock a 4 ° C durante 30 minutos. Algunos sugirieron que los volúmenes de tampón de lisis para una variedad de tamaños de placas: <ul><li> 6-así placa de 0,5 ml por pozo </li><li> 10 cm placa de 1 ml por placa </li><li> 15 cm placa de 1 ml por placa </li></ul></li><li> Después de la lisis, utilice un rascador de células para recoger las células de la placa. Asegúrese de mantener lisados ​​en hielo para minimizar la actividad de la proteasa </li><li> Lisados ​​celulares girar durante 15 minutos a 4 ° C en una sobremesa mini-centrífuga a toda velocidad (15.000 xg). </li><li> Pipeta el sobrenadante a un tubo nuevo. Deseche el sedimento. </li><li> Analizar los niveles de proteína y proceder con la inmunoprecipitación. </li></ol> 6. Preparación de inmuno-magnética bolas Precipitaciones Nota: Este paso se suele iniciar directamente después del inicio de la incubación de entrecruzamiento de 2 horas. <ol><li> Combine 30 l de Dynal bolas inmunomagnéticas, 500 l de tampón IP (1x tampón A + 0,1% Triton X-100) y la cantidad apropiada de antígeno-anticuerpo específico de rosca para tubos de microcentrífuga. Prepare libre de anticuerpos y no específica de anticuerpos para los tubos de los controles negativos. La cantidad apropiada de anticuerpos se determina en base a los antígenos en experimentos separados. Marque todos los tubos de propiedad intelectual. </li><li> Permita que los tubos de IP para la incubación de un rockero de extremo a extremo a temperatura ambiente durante dos horas. Por otra parte, incubar los tubos IP con anticuerpos noche a 4 ° día antes de la reticulación. </li><li> Después de la incubación, hacer una rápida 10 segundos mini-centrifugación para recoger las gotas en la parte inferior del tubo. </li><li> Deslice los tubos en el soporte magnético, que se tire de la partículas magnéticas de la parte inferior del tubo. Con las cuentas de forma segura fuera del camino, ahora puede fácilmente eliminar cualquier anticuerpo no unido. </li><li> Use una pequeña propina aspirador dio como una punta de carga de gel, o p200 punta para quitar el tampón de incubación y cualquier anticuerpo no unido. Tan pronto como el volumen de incubación se retira en añadir 1 mL de tampón IP. </li><li> Tapar el tubo, con cuidado volver a suspender las cuentas, e incubar todos los tubos de IP a temperatura ambiente durante 5 minutos con final sobre el extremo de rotación. </li><li> Repita el paso de lavado de 5 minutos (pasos 6.3 a 6.6), una vez más con un mililitro de tampón fresco IP. </li><li> Los tubos IP puede permanecer en la incubación, lavar durará hasta el lisado reticulado está listo para ser añadido a las perlas. </li></ol> 7. Incubar lisado reticulado con inmuno-magnética Cuentas <ol><li> Después del último lavado de giro inmuno-magnética cuentas, en un mini-centrifugar durante 10 segundos. A continuación, proceder a caer los tubos en el soporte magnético. </li><li> Una vez más, con una pequeña propina aspiración agujero, quitar el volumen de incubación del tubo. Inmediatamente añadir 500 l de lisado celular reticulado (suponiendo lisado a 1 mg / ml) a los tubos que contienen inmuno-magnética cuentas. Si va a utilizar la competencia de péptidos como control negativo, una cantidad adecuada de péptido se debe incluir en este punto. Los experimentos anteriores se debe realizar para determinar las condiciones de competencia adecuadas péptido. Alternativamente, usted puede querer tener lisado libre inmuno-magnética cuentas como control negativo. En este caso, inmediatamente añadir 500 l de lisado buffer (1x tampón A + 0,5% Triton X-100) + completa de las cuentas de inmuno-magnética. </li><li> Resuspender suavemente las bolas. Microesferas resuspendidas se incubaron a 4 º en una rotación de extremo a extremo durante 2 horas. </li></ol> 8. Lave lisado consolidar de Cuentas <ol><li> Una vez lisado ha tenido tiempo de incubación suficiente, hace un rápido giro 10 segundos en un mini-centrifugadora. A continuación, deslice los tubos en el soporte magnético. El soporte magnético se debe mantener en un baño de hielo durante el resto del experimento. </li><li> Usando una pequeña punta del aspirador dio, quite todas las lisado no consolidados. Inmediatamente después de todo lisado no unido se elimina añadir 1 ml de tampón de IP. </li><li> Tape el tubo y suavemente volver a suspender las bolas. </li><li> Cuando los granos se resuspendió repetir el paso de granulación de cuentas (8,1). </li><li> Una vez más aspirado de todo el volumen de incubación y de inmediato agregar 1 ml de buffer de propiedad intelectual, a continuación, tapar el tubo. </li><li> Resuspenda suavemente los granos en el tampón de IP. Incubar microesferas resuspendidas durante 5 minutos a 4 ° con el de extremo a extremo balanceo. </li><li> Repita los pasos de lavado de grano (8.4 a 8.6) 4 veces más. </li></ol> 9. Desnaturalizar la muestra y la Colecta de Cuentas <ol><li> Después de todos los tubos de precipitación inmuno-magnética han sido lavados, sedimentos volver a las cuentas (8,1) y las trompas de caer en lasoporte magnético. </li><li> Proteínas unidas a las perlas inmuno-magnéticas se eliminan en condiciones de desnaturalización. Aspirar fuera del lavado IP Buffer pasado y retirar el tubo de IP del soporte magnético. </li><li> Añadir 1x tampón de la muestra de gel en el tubo de IP justo por encima de la pastilla a la piscina de bolas las cuentas en la parte inferior del tubo de nuevo. La cantidad de tampón de la muestra depende del tamaño y del gel que se utiliza. Es posible que deba golpear suavemente la parte inferior del tubo para obtener todas las cuentas se resuspendieron en el tampón de la muestra de gel. </li><li> Repita el proceso de desnaturalización (9,2 por 9,3) para cada tubo IP. </li><li> Inmuno-magnética microesferas resuspendidas en tampón de gel de la muestra se calienta a 75 ° C durante 5 minutos para completar el proceso de desnaturalización. </li><li> Una vez que la muestra ha sido desnaturalizada que se puede ejecutar en un gel de SDS-PAGE para el Western Blot. Vaciado Inmuno-Magnética bolas se puede volver a sedimentaron por centrifugación y se retira de la muestra con el soporte magnético. </li></ol> 10. Elución de los complejos entrecruzados con péptidos antigénicos (cromatografía de inmunoafinidad). <ol><li> Después de todos los tubos de inmunoprecipitación inmuno-magnética han sido lavados, sedimentos volver a las cuentas (8,1) y las trompas de diapositiva en el soporte magnético. Aspirar el sobrenadante y añadir 10 l de tampón A complementado con el péptido antigénico reconocido por el anticuerpo utilizado para immunoisolation de complejos de proteínas. Usted debe probar concentraciones que van desde 10 hasta 200 M para elución eficiente. </li><li> Incubar las cuentas durante 2 horas a 4 ° C. </li><li> Poner cuentas en el soporte magnético y con mucho cuidado recuperar los 10 ml de material eluido. Guardar este sobrenadante. </li><li> Lave rápidamente las cuentas con un Buffer y deseche el lavado. Salvar las cuentas. </li><li> Añadir Buffer Gel de muestra para guardar el sobrenadante y cuentas a una concentración de 1x. Incubar los tubos a 75 ° C durante 5 minutos. </li><li> Analizar cuentas y eluido péptido por SDS-PAGE. Que el efluente debe estar libre de IgG, tanto por tinción de proteínas de la SDS-PAGE o por immunoblots (fig. 1B). Este material libre de IgG es adecuado para la espectrometría de masas. </li></ol><img src="/files/ftp_upload/1855/1855fig1.jpg" alt="Figura 1" /> Figura 1. El aislamiento de AP-3 complejos de proteínas que interactúan y proteínas de la membrana. HEK293 (A) o las células PC12 (B) fueron tratados en la presencia de control del vehículo (DMSO, extraño carriles Fig. 1A) o DSP (incluso carriles fig. 1A o todos los carriles en la Fig. 1B). Extractos se incubaron ya sea aclarado con cuentas solo (Fig. 1A, carriles 3-4), los anticuerpos del receptor de transferrina (Fig. 1A, carriles 5-6), o AP-3 anticuerpos δ (Fig. 1A, carriles 7-10; Fig. . 1B, carriles 1-10). Los complejos inmunes se eluyeron con tampón de muestra SDS-PAGE (Fig. 1A), un tampón solo o (Fig. 1B, carriles 1-2), o un tampón suplementado con concentraciones crecientes del péptido antigénico δ correspondientes a los aminoácidos 680 710 de los derechos humanos δ-adaptin (NCBI: AAD03777; GI: 1923266) (Fig. 1B, carriles 30-10). Este péptido se une al anticuerpo δ. Tras la elución de péptidos, los sobrenadantes (S) y cuentas (B) se analizaron por inmunoblot (Fig. 1B). AP-3, que se detecta con anticuerpos contra el δ, β3, y las subunidades σ3, coprecipitados con los siguientes factores solubles: la cadena pesada de clatrina (CHC), del BLOC-1 pallidin subunidad, y la subunidad vps33b LÚPULO complejo, así como la membrana de proteínas fosfatidilinositol-4-quinasa tipo II alfa (PI4KIIα) y el transportador de zinc 3 (ZnT3). Nótese la ausencia de IgG de ratón cadenas pesadas en el péptido eluido sobrenadante de la figura. 1B.

<ol>
	<li>Salazar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hermansky-Pudlak+syndrome+protein+complexes+associate+with+phosphatidylinositol+4-kinase+type+II+alpha+in+neuronal+and+non-neuronal+cells.">Hermansky-Pudlak syndrome protein complexes associate with phosphatidylinositol 4-kinase type II alpha in neuronal and non-neuronal cells.</a> J Biol Chem. 284, 1790-1802 (2009).</li><li>Lomant, A. J., Fairbanks, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+probes+of+extended+biological+structures:+synthesis+and+properties+of+the+cleavable+protein+cross-linking+reagent+[35S]dithiobis(succinimidyl+propionate.">Chemical probes of extended biological structures: synthesis and properties of the cleavable protein cross-linking reagent [35S]dithiobis(succinimidyl propionate.</a> J Mol Biol. 104, 243-261 (1976).</li><li>Xiang, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+DSP,+a+reversible+cross-linker,+to+fix+tissue+sections+for+immunostaining,+microdissection+and+expression+profiling.">Using DSP, a reversible cross-linker, to fix tissue sections for immunostaining, microdissection and expression profiling.</a> Nucleic Acids Res. 32, e185-e185 (2004).</li><li>Craige, B., Salazar, G., Faundez, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylinositol-4-Kinase+Type+II+Alpha+Contains+an+AP-3+Sorting+Motif+and+a+Kinase+Domain+that+are+both+Required+for+Endosome+Traffic.">Phosphatidylinositol-4-Kinase Type II Alpha Contains an AP-3 Sorting Motif and a Kinase Domain that are both Required for Endosome Traffic.</a> Mol Biol Cell. 19, 1415-1426 (2008).</li><li>Newell-Litwa, K., Seong, E., Burmeister, M., Faundez, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+and+non-neuronal+functions+of+the+AP-3+sorting+machinery.">Neuronal and non-neuronal functions of the AP-3 sorting machinery.</a> J Cell Sci. 120, 531-541 (2007).</li><li>Salazar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zinc+Transporter+ZnT3+interacts+with+AP-3+and+it+is+Targeted+to+a+Distinct+Synaptic+Vesicle+Subpopulation.">The Zinc Transporter ZnT3 interacts with AP-3 and it is Targeted to a Distinct Synaptic Vesicle Subpopulation.</a> Mol Biol Cell. 15, 575-587 (2004).</li><li>Trinkle-Mulcahy, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+specific+protein+interaction+partners+using+quantitative+mass+spectrometry+and+bead+proteomes.">Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes.</a> J Cell Biol. 183, 223-239 (2008).</li></ol>

DSP, una membrana permeable, reticulante químicamente reduce con un brazo espaciador de 12 A se utiliza para estabilizar las interacciones proteína transitoria 1,2,3,4. Aquí se ejemplifican esta estrategia con el complejo adaptador AP-3 un complejo de proteínas solubles, que reconoce y clasifica las proteínas de membrana en vesículas de endosomas 5. AP-3 se une selectivamente al transportador de zinc ZnT3 y la quinasa de lípidos fosfatidilinositol-4-quinasa alfa de tipo II, pero no del recep...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
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<td>Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P4417</td>
<td>Dissolve 1 tablet in 200 mL water; add MgCl2 to a final concentration of 1 mM and CaCl2 to a final concentration of 0.1 mM</td>
</tr>
<tr>
<td>Dithiobis (succinimidyl propionate) (DSP)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>22585</td>
<td>Moisture sensitive, store in air tight container 4&deg;C</td>
</tr>
<tr>
<td>Dimethyl sulphoxide (DMSO) Hybri-Max</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D2650</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100, SigmaUltra</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T9284</td>
<td>&nbsp;</td>
<tr>
<td>Dynabeads, Sheep anti-Mouse IgG</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>110.31</td>
<td>Beads are also available as sheep anti-rabbit</td>
</tr>
<tr>
<td>Dyna-Mag-2 magnet</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>123-21D</td>
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isolation of labile multi-protein complexes by in vivo controlled cellular cross-linking and immuno-magnetic affinity chromatography

La naturaleza dinámica de los mecanismos celulares con frecuencia se basa en las asociaciones de proteínas transitoria y / o débiles. Estas interacciones de baja afinidad excluye métodos estrictos para el aislamiento e identificación de las redes de proteínas alrededor de una proteína de interés. El uso de productos químicos reticulantes permite la estabilización selectiva de las interacciones lábiles, evitando así las limitaciones de bioquímica para la purificación. Aquí se presenta un protocolo susceptible de células en cultivo que utiliza un agente de reticulación homobifuncionales con un brazo espaciador de 12 Å, ditiobis (propionato de succinimidilo) (DSP). DSP se separa por reducción de un puente disulfuro en la molécula. La reticulación en combinación con la cromatografía de inmunoafinidad de proteínas de interés con bolas magnéticas permite el aislamiento de complejos de proteínas que de otro modo no resistiría la purificación. Este protocolo es compatible con las técnicas regulares de Western blot y puede ser ampliado para la identificación de proteínas por espectrometría de masas 1. Stephanie A. Zlatic y Pearl V. Ryder contribuyeron igualmente a este trabajo.

Watch this Scientific Journal Video about Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography at JoVE.com

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

La célula permeable reticulante DSP [ditiobis (propionato de succinimidilo)] estabiliza interacciones transitoria e inestable<em&gt; In vivo</em&gt;, Lo que permite su aislamiento con estrictas técnicas de purificación de proteínas complejas. Aquí se presenta una técnica para que las células en un cultivo de entrecruzamiento seguido por el aislamiento de complejos de proteínas por inmunoprecipitación.

El aislamiento de lábil Multi-complejos de proteínas por In vivo Control Celular entrecruzamiento y Inmuno-cromatografía de afinidad magnética

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

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Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

17.2K vistas.  Emory University. La célula permeable reticulante DSP [ditiobis (propionato de succinimidilo)] estabiliza interacciones transitoria e inestable In vivo, Lo que permite su aislamiento con estrictas técnicas de purificación de proteínas complejas. Aquí se presenta una técnica para que las células en un cultivo de entrecruzamiento seguido por el aislamiento de complejos de proteínas por inmunoprecipitación.

Video: Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, Parkinson's, and Huntington's diseases, hereditary amyotrophic lateral sclerosis, and type 2 diabetes. Owing to the metastable nature of these protein assemblies, it is difficult to assess their oligomer size distribution quantitatively using classical methods, such as electrophoresis, chromatography, fluorescence, or dynamic light scattering. Oligomers of amyloidogenic proteins exist as metastable mixtures, in which the oligomers dissociate into monomers and associate into larger assemblies simultaneously. PICUP stabilizes oligomer populations by covalent cross-linking and when combined with fractionation methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or size-exclusion chromatography (SEC), PICUP provides snapshots of the oligomer size distributions that existed before cross-linking. Hence, PICUP enables visualization and quantitative analysis of metastable protein populations and can be used to monitor assembly and decipher relationships between sequence modifications and oligomerization1. Mechanistically, PICUP involves photo-oxidation of Ru2+ in a tris(bipyridyl)Ru(II) complex (RuBpy) to Ru3+ by irradiation with visible light in the presence of an electron acceptor. Ru3+ is a strong one-electron oxidizer capable of abstracting an electron from a neighboring protein molecule, generating a protein radical1,2. Radicals are unstable, highly-reactive species and therefore disappear rapidly through a variety of intra- and intermolecular reactions. A radical may utilize the high energy of an unpaired electron to react with another protein monomer forming a dimeric radical, which subsequently loses a hydrogen atom and forms a stable, covalently-linked dimer. The dimer may then react further through a similar mechanism with monomers or other dimers to form higher-order oligomers. Advantages of PICUP relative to other photo- or chemical cross-linking methods3,4 include short (&#8804;1 s) exposure to non-destructive visible light, no need for pre facto modification of the native sequence, and zero-length covalent cross-linking. In addition, PICUP enables cross-linking of proteins within wide pH and temperature ranges, including physiologic parameters. Here, we demonstrate application of PICUP to cross-linking of three amyloidogenic proteins the 40- and 42-residue amyloid &beta;-protein variants (A&beta;40 and A&beta;42), and calcitonin, and a control protein, growth-hormone releasing factor (GRF).

Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides

Photo-induced cross-linking of unmodified proteins (PICUP) allows characterization of oligomer size distribution in metastable protein mixtures. We demonstrate application of PICUP to three representative amyloidogenic peptides the 40- and 42-residue forms of amyloid &beta;-protein, and calcitonin, and a control peptide growth-hormone releasing factor.

Foto-inducido el entrecruzamiento de las proteínas sin modificar (picup) aplicada a los péptidos amiloidogénicas

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, ...

Investigación

Biología

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Identificación de los complejos de proteínas con la proteómica cuantitativa en S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

Visualizar solo los complejos moleculares En Vivo Uso de microscopía de fluorescencia avanzada

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

ELIME (Enzyme Linked Inmuno magnética químicos) Método para la detección de micotoxinas

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

Imágenes in vivo de larvas de Drosophila intactos a nivel sub-celular Resolución

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.

A general protocol for the use of isothermal titration calorimetry to monitor the binding thermodynamics for biological systems with moderate binding affinities is presented.

Calorimetría de titulación isotérmica para la medición de macromoléculas-ligando de afinidad

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction.  In biological ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Aislamiento de la cromatina mediante la purificación de ARN (chirp)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Identificación de los complejos de proteínas en<em&gt; Escherichia coli</em&gt; La purificación mediante afinidad del péptido secuencial en combinación con espectrometría de masas tándem

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

Cartográficas basadas en AFM de las propiedades elásticas de las paredes celulares: en tejido, celular, y las Resoluciones Subcelulares

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

En Vivo Tracking 4-Dimensional de madre hematopoyéticas y células progenitoras en ratones adultos de calota de Médula Ósea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...