Nos gustaría dar las gracias lo siguiente para apoyo financiero: BBSRC Instituto de Financiación de Programas Estratégicos - CF &amp; TMW; Financiación BBSRC Oriente Bio DTP - LG; El Darwin Fideicomiso de Edimburgo - MLH. También nos gustaría dar las gracias a Dr. Barry McColl permiso para incluir la optimización TREM2 en este manuscrito.

<ol>
	<li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Biotechnology. 1992 (24), 145-149 (1979).</li><li>Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protein Protocols on CD-ROM. , (1998).</li><li>Gallagher, S., Winston, S. E., Fuller, S. A., Hurrell, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoblotting+and+immunodetection.">Immunoblotting and immunodetection.</a> Curr Protoc Mol Biol. Chapter. 10, 10 (2008).</li><li>Eaton, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+protein+analysis+as+a+reliable+loading+control+for+quantitative+fluorescent+Western+blotting.">Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting.</a> PLoS One. 8 (8), 72457 (2013).</li><li>Suzuki, O., Koura, M., Noguchi, Y., Uchio-Yamada, K., Matsuda, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+sample+mixtures+for+standard+curve+creation+in+quantitative+Western+blots.">Use of sample mixtures for standard curve creation in quantitative Western blots.</a> Exp. Anim. 60, 193-196 (2011).</li><li>Colella, A. D., 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=Comparison+of+Stain-Free+gels+with+traditional+immunoblot+loading+control+methodology.">Comparison of Stain-Free gels with traditional immunoblot loading control methodology.</a> Anal Biochem. 430 (2), 108-110 (2012).</li><li>Zellner, M., 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=Fluorescence-based+Western+blotting+for+quantitation+of+protein+biomarkers+in+clinical+samples.">Fluorescence-based Western blotting for quantitation of protein biomarkers in clinical samples.</a> Electrophoresis. 29, 3621-3627 (2008).</li><li>Kielar, 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=Molecular+correlates+of+axonal+and+synaptic+pathology+in+mouse+models+of+Batten+disease.">Molecular correlates of axonal and synaptic pathology in mouse models of Batten disease.</a> Hum Mol Genet. 18, 4066-4080 (2009).</li><li>Mutsaers, C. A., Lamont, D. J., Hunter, G., Wishart, T. M., Gillingwater, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+proteomics+identifies+Calreticulin+and+GRP75/Mortalin+as+peripherally+accessible+protein+biomarkers+for+spinal+muscular+atrophy.">Label-free proteomics identifies Calreticulin and GRP75/Mortalin as peripherally accessible protein biomarkers for spinal muscular atrophy.</a> Genome Med. 5 (10), 95 (2013).</li><li>Wishart, T. M., 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=Dysregulation+of+ubiquitin+homeostasis+and+&#946;-catenin+signaling+promote+spinal+muscular+atrophy.">Dysregulation of ubiquitin homeostasis and &#946;-catenin signaling promote spinal muscular atrophy.</a> J Clin Invest. 124 (4), 1821-1834 .</li><li>Mathews, S. T., Plaisance, E. P., Kim, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+systems+for+westerns:+chemiluminescence+vs.+infrared+detection.">Imaging systems for westerns: chemiluminescence vs. infrared detection.</a> Methods Mol Biol. 536, 499-513 (2009).</li><li>Hawes, J. J., Brunzell, D. H., Wynick, D., Zachariou, V., Picciotto, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GalR1,+but+not+GalR2+or+GalR3,+levels+are+regulated+by+galanin+signalling+in+the+locus+coeruleus+through+a+cyclic+AMP-dependent+mechanism.">GalR1, but not GalR2 or GalR3, levels are regulated by galanin signalling in the locus coeruleus through a cyclic AMP-dependent mechanism.</a> J Neurochem. 93 (5), 1168-1176 (2006).</li><li>Bond, D., Primrose, D. A., Foley, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+evaluation+of+signaling+events+in+Drosophila+S2+cells.">Quantitative evaluation of signaling events in Drosophila S2 cells.</a> Biol Proceed Online. 10 (1), 20-28 (2008).</li><li>Wishart, T. M., 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=Differential+proteomics+analysis+of+synaptic+proteins+identifies+potential+cellular+targets+and+protein+mediators+of+synaptic+neuroprotection+conferred+by+the+slow+Wallerian+degeneration+(Wlds)+gene.">Differential proteomics analysis of synaptic proteins identifies potential cellular targets and protein mediators of synaptic neuroprotection conferred by the slow Wallerian degeneration (Wlds) gene.</a> Mol Cell Proteomics. 6, 1318-1330 (2007).</li><li>Silva, J. M., McMahon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fastest+western+in+town:+a+contemporary+twist+on+the+classic+western+blot+analysis.">The fastest western in town: a contemporary twist on the classic western blot analysis.</a> J. Vis. Exp. (84), 51149 (2014).</li></ol>

The authors have nothing to disclose.

La debida consideración y la planificación es esencial antes de cualquier experimento y en última instancia pueden determinar el éxito de la técnica utilizada. Los avances en la detección de proteínas utilizando WB puede presentar una gran cantidad de posibles obstáculos cuando se trata de elegir los anticuerpos, métodos de transferencia y visualización adecuadas para su uso. Afortunadamente, el uso de una lista de verificación cuidadosa y medidas de control apropiadas QFWB puede ser utilizado de forma rutinaria para determinar la presencia de proteínas y las diferencias de expresión cada vez más sutiles entre las muestras. Este protocolo ofrece una guía completa sobre el Western Blot cuantitativa fluorescente, así como algunas estrategias de solución de problemas para evitar y / o resolver algunos de los muchos errores comunes asociados a ella. Los pasos críticos usadas para mantener la sensibilidad y obtener verdaderamente mediciones cuantificables y comparables incluyen: 1) robusta extracción de proteína a partir de muestras de tejido; 2) preparación de la muestra; 3) loadin proteína exactag determinó por análisis de proteína total; 4) la transferencia óptima de proteínas utilizando I-Blot; 5) la preparación de anticuerpos primarios y secundarios en tampón de bloqueo que contiene 0,1% de Tween 20, y 6) correcta visualización y análisis utilizando un generador de imágenes de infrarrojos y el software asociado. Detección fluorescente infrarrojo es verdaderamente cuantitativo y proporciona una mayor sensibilidad en comparación con las técnicas más tradicionales de detección ECL 3,11. Este sistema de detección es de múltiples facetas, y como tal no se limita a QFWB. Este sistema es capaz de obtención de imágenes de etiquetado inmunohistoquímico en baja potencia que permite la visualización y cuantificación de las secciones de tejido 12 enteros. Esta es un área de potencial de desarrollo futuro en términos de resolución, que podría ver imágenes rojo lejano que rivaliza con la captura de inmunofluorescencia convencional con microscopios convencionales en términos de evaluación cuantitativa. Sin embargo, con una mayor sensibilidad a los cambios sutiles en protein expresión es fundamental para garantizar la variabilidad se reduce al mínimo y las medidas de control son muy estrictos con los protocolos robustos. Esto comienza con la extracción de proteínas rigurosa de la muestra de tejido seguido por la producción de geles de proteínas totales se tiñeron para proporcionar la seguridad de que la carga de muestra es uniforme, la optimización de anticuerpos primarios y secundarios para determinar si la detección es real y prueba de las directrices del fabricante con respecto a la transferencia veces para obtener la proteína de transferencia eficiente. Sin embargo, incluso cuando se optimizan las condiciones para WB, es posible que aún los problemas asociados con el funcionamiento de los westerns que pueden no haber sido plenamente exploradas aquí. Estos incluyen, pero no se limitan a factores que incluyen la solubilización de proteínas y la elección de tampón de extracción. Algunos tampones pueden interferir con ensayos de concentración de proteína, y algunos tejidos son particularmente difíciles de solubilizar, lo que requiere técnicas más robustas tales como el uso de macera automatizadoting envases sellados, tales como tubos de M junto con un disociador Macs. Además, las medidas de control sencillas para el almacenamiento de material extraído no extraídos y a -80 ° C pueden ser la diferencia entre la obtención de etiquetado óptimo inmediatamente después de la extracción y que tiene resultados pobres semanas más tarde. Métodos QFWB modernos han demostrado ser más sensibles para captar las diferencias sutiles en la expresión de proteínas y están permitiendo que más versátil de doble etiquetado simultáneo de 3, en comparación con las técnicas más antiguas como la ECL. Es de vital importancia que los protocolos de Western Blot son robustos y fácilmente repetible para la cuantificación precisa y el análisis estadístico. Este protocolo es sensible y lo suficientemente robusto como para ser utilizado de forma rutinaria para la detección de proteínas a través de una variedad de diferentes muestras de tejidos y especies 3 y permite la cuantificación de las proteínas de baja y alta abundancia dentro de la misma QFWB por lo tanto reduciendo el uso de consumibles así como el tiempo por experimento 1. 1 Además, el aumento de la sensibilidad de esta técnica permite la validación de cada vez más popular - estudios ómicas 9,14 embargo exactitud es crucial y la inclusión de medidas de control apropiadas deben ser respetados evitando de este modo la adquisición de datos erróneos.

Western Blot (WB) es una técnica analítica desarrollado originalmente en la década de 1970 para determinar la presencia o ausencia de una proteína de interés en una muestra biológica compleja, tal como un homogeneizado de tejido 1. Comúnmente conocido como la inmunotransferencia de proteínas, debido a la interacción anticuerpo-antígeno clave, la metodología consiste en 5 pasos distintos: 1) la separación electroforética de las proteínas por su punto isoeléctrico; 2) la transferencia a un nitrocelulosa o difluoruro de polivinilideno (PVDF) membrana; 3) marcar usando un anticuerpo primario específico para la proteína de interés; 4) la incubación con un anticuerpo secundario dirigido contra el anticuerpo primario; y 5) la visualización. Visualización metodología ha evolucionado con el tiempo para mejorar la seguridad y la sensibilidad. Algunos de los primeros WB se llevaron a cabo utilizando etiquetas de radio etiquetadas que luego progresaron a los métodos quimioluminiscentes más ampliamente utilizado (ECL) colorimétrico y luego. RadioactiviTy se marcó directamente en sondas para antígenos específicos mientras que las metodologías colorimétricas y ECL utilizan una técnica indirecta etiquetado con una enzima tal como reportero alcalina fosfatasa o estreptavidina peroxidasa de rábano picante (HRP) 2. La intensidad de etiquetado del producto cromógeno o luminiscente se mide utilizando densitometría por lo que la intensidad de la señal, fuerte o débil, más o menos indica la presencia de la proteína de interés en la muestra. ECL es la metodología más sensible y, por tanto, favorecido 2, pero los 3 métodos se desarrollaron inicialmente en la película de rayos X con técnicas de imagen digital más sofisticados posteriormente establecidas 3. El avance de la imagen digital de WB no sólo permite a un investigador para determinar la presencia o ausencia de su proteína de interés, pero también permitió una inferencia con el nivel de expresión de una proteína seleccionada cuando se compara con otras muestras y por lo tanto puede ser denominado &quot;semi-cuantitativa &amp; #8221 ;. Recientemente, una tecnología de transferencia de western verdaderamente cuantitativo y más sensibles ha sido desarrollado por el cual el nivel de fluorescencia medido está directamente relacionada con la cantidad y la expresión de una sola proteína dentro de una muestra: cuantitativa fluorescente Western Blot (QFWB). Al comparar con el etiquetado QFWB ECL, el uso de un anticuerpo secundario fluorescente genera un perfil de detección lineal 4. Esto está en contraste con las técnicas ECL donde la linealidad de la señal generalmente se produce con cargas bajas de proteínas por debajo de 5 mg y la saturación de señal directamente relacionados con la expresión de la proteína, es decir, con genes de limpieza ubicuamente expresadas 5,6. Esta disparidad es muy probablemente causado por un mayor número de sitios de unión disponibles para un sustrato ECL avidina para unirse a un secundario biotinilado, que resulta en una mayor probabilidad de potencial de saturación de la señal. Esta es una de las principales razones de inmunotransferencia ECL base que se conoce como only &quot;semi-cuantitativa&quot; 7. El punto de saturación de la señal es de importancia crítica en la medición de las diferencias sutiles en los niveles de expresión y puede dar lugar a mediciones inexactas. En los últimos años el advenimiento de técnicas proteómicas generalizadas detallando cada vez mayor sensibilidad y la identificación de las diferencias sutiles de expresión ha dado lugar a una confianza cada vez mayor en la transferencia Western cuantitativa verdaderamente para los experimentos de validación de 8,9. La aplicación de la metodología sensible, robusto y verdaderamente cuantitativo es por lo tanto crucial. Muchas metodologías Western Blot &quot;optimizados&quot; han sido utilizados por los laboratorios independientes, que con frecuencia resultan difíciles de establecer o reproducir debido a los ajustes metodológicos sutiles que pueden no estar reflejados en los protocolos documentados formales. Se trata de un protocolo establecido y robusto para QFWB y, además, ofrece valiosas estrategias para solucionar problemas comunes that puedan surgir durante la ejecución. Este protocolo se optimizó originalmente para su uso con homogenados de cerebro murino, pero desde entonces ha sido utilizado con eficacia en una amplia gama de muestras de tejidos y especies 4,9,10. Variaciones del protocolo potenciales necesarios para la solución de problemas específicos se incluyen.

<table border="0" cellpadding="0" cellspacing="0" width="1024">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:427px;">Name of Material/ Equipment</td>
			<td style="width:255px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:224px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">RIPA Buffer</td>
			<td style="width:255px;">Fisher Scientific UK Ltd</td>
			<td>10230544</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">M Tubes</td>
			<td style="width:255px;">Miltenyi Biotec Inc.</td>
			<td>130-093-236</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:427px;">iBlot Transfer Stack, PVDF Regular</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td>IB401001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">MagicMark XP Western Protein Standard (20-220 kDa)</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td>LC5602</td>
			<td>Use in gel 1 for Western blotting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">SeeBlue Pre-stained protein standard</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td>LC5625&#160;</td>
			<td>Use in gel 2 Total protein stained gel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">NuPAge LDS Sample buffer 4X</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td>NP0007</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:427px;">NuPAGE MES SDS Running Buffer (for Bis-Tris Gels only) (20X)</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td>NP0002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">NuPAGE Novex 4-12% Bis-Tris Gel 1.0 mm, 12 well</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td>NP0322BOX</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:427px;">PHOSPHATE BUFFERED SALINE TABLET,*TRU-ME, PHOSPHATE BUFFERED SALINE TABLET</td>
			<td style="width:255px;">Sigma-Aldrich, UK</td>
			<td>P4417-100TAB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Micro BCA, Protein Assay Kit</td>
			<td style="width:255px;">Fisher Scientific UK Ltd</td>
			<td>10249133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Odyssey&#160; blocking buffer</td>
			<td style="width:255px;">Li-Cor Biosciences</td>
			<td>P/N 927-40000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">IRDye 680RD Goat anti-Rabbit IgG (H+L), 0.5 mg</td>
			<td style="width:255px;">Li-Cor Biosciences</td>
			<td style="width:119px;">926-68071</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">IRDye 680RD Donkey anti-Mouse IgG (H+L), 0.5 mg&#160;</td>
			<td style="width:255px;">Li-Cor Biosciences</td>
			<td style="width:119px;">926-68072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">IRDye 800CW Goat anti-Rabbit IgG (H + L), 0.5mg</td>
			<td style="width:255px;">Li-Cor Biosciences</td>
			<td style="width:119px;">926-32211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">IRDye 800CW Donkey anti-Goat IgG (H + L), 0.5mg</td>
			<td style="width:255px;">Li-Cor Biosciences</td>
			<td style="width:119px;">926-32214</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">ODYSSEY CL Infra-red imager</td>
			<td style="width:255px;">Li-Cor Biosciences</td>
			<td style="width:119px;"></td>
			<td>Call for quotation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">iBlot 7-Minute Blotting System</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td style="width:119px;"></td>
			<td>This model is no longer in production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">InstantBlue Protein stain</td>
			<td style="width:255px;">Expedeon, UK</td>
			<td>ISB1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Revitablot western blot stripping buffer&#160;</td>
			<td>Rockland Immunochemicals Inc.</td>
			<td>MB-085-0050</td>
			<td></td>
		</tr>
	</tbody>
</table>

a guide to modern quantitative fluorescent western blotting with troubleshooting strategies

Los finales de los años 1970 vio el primer uso informado públicamente de la transferencia de western, una técnica para evaluar la presencia y abundancia relativa de las proteínas específicas dentro de muestras biológicas complejas. Desde entonces, la metodología de transferencia de western se ha convertido en un componente común de los biólogos moleculares el repertorio experimental. Una búsqueda superficial de PubMed utilizando el término &quot;western blot&quot; sugiere que más de doscientos veinte mil manuscritos publicados han hecho uso de esta técnica para el año 2014. Es importante destacar que, en los últimos diez años se ha producido avances técnicos de imagen, junto con el desarrollo marcadores fluorescentes de sensibles que han mejorado la sensibilidad y arrojado aún mayores rangos de detección lineal. El resultado es una fluorescencia ahora verdaderamente cuantificable basado Western Blot (QFWB) que permite a los biólogos para llevar a cabo el análisis de expresión comparativo con mayor sensibilidad y precisión que nunca antes. Muchos Western Blot &quot;optimizado&quot;Existen metodologías y se utilizan en diferentes laboratorios. Estos a menudo resultan difíciles de implementar debido a la exigencia de modificaciones procesales sutiles pero indocumentados. Este protocolo proporciona una descripción completa de un método QFWB establecido y robusto, completo con las estrategias de solución de problemas.

Como la sensibilidad QFWB y el rango lineal de detección es mayor que la detección convencional ECL, hay una serie de medidas de control que son cruciales para asegurar la recepción de datos precisa, ayudando de ese modo la interpretación efectiva. En primer lugar, la inclusión de muestras de control positivas como se muestra en la Figura 1. En segundo lugar, la optimización de la transferencia para garantizar una circulación equivalente de proteínas de alto y bajo peso molecular desde el gel a la membrana según lo exhibido en la figura 2. En tercer lugar, la optimización de anticuerpos, especialmente secundaria anticuerpos cuya optimización a menudo se pasa por alto, pero que pueden producir no específica bandas capaces de interferir con la interpretación correcta de la proteína (s) de interés. Véase la Figura 3. En cuarto lugar, también puede ser el caso cuando una proteína que aparece indetectable pero se espera que esté presente, esto también puede ser un problema de anticuerpo secundario que se puede corregir utilizando simplemente un rais secundariased en una serie de especies diferentes. Véase la Figura 4. En quinto lugar, el etiquetado de proteína total y el análisis es un método mucho más robusto y cuantificable en comparación con el uso de la proteína simple tradicional (s) que se expresa de forma ubicua para los estándares de referencia internos 3. Muchas de estas proteínas individuales se han encontrado para ser expresadas diferencialmente en modelos de enfermedades neurodegenerativas, así como entre las diferentes muestras de tejido y la uniformidad de expresión puede modificar dentro del mismo tejido 3. Por lo tanto, la producción de un gel de control de carga confirmará la uniformidad de la carga de la muestra cuando se combina con un análisis de proteína total mediante la comparación y la cuantificación de la carga de proteína en cada carril en varios rangos de pesos moleculares medidos contra cada muestra para indicar el error estándar como se demuestra en la Figura 6 . Es importante destacar que todas estas técnicas y controles de solución de problemas sólo son tan eficaces como la sensibilidad y la coherencia del análisis terramientas aplicadas por el operador (Figura 5). Finalmente esta técnica se presta a la extracción y re-sondeo de las membranas con más flexibilidad que ECL debido a factores incluyendo, pero no limitado a aumento de la sensibilidad, la reducción de fondo, la detección de doble color y estabilidad de la membrana en condiciones de almacenamiento a largo plazo. Véase la Figura 7.

Watch this Scientific Journal Video about A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies at JoVE.com

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

Una guía para la moderna cuantitativa fluorescente Western Blot con las estrategias de solución de problemas

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

a-guide-to-modern-quantitative-fluorescent-western-blotting-with

Research

JoVE Journal

Biology

50.9K vistas.  University of Edinburgh. The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

Video: A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the relative amount of the protein present in different samples, and analyze the results of co-immunoprecipitation experiments. In this method, a target protein is detected with a specific primary antibody in a given sample of tissue homogenate or extract. Protein separation according to molecular weight is achieved using denaturing SDS-PAGE. After transfer to a membrane, the target protein is probed with a specific primary antibody and detected by chemiluminescence.
Since its first description, the western-blotting technique has undergone several improvements, including pre-cast gels and user-friendly equipment. In our laboratory, we have chosen to use the commercially available NuPAGE electrophoresis system from Invitrogen. It is an innovative neutral pH, discontinuous SDS-PAGE, pre-cast mini-gel system. This system presents several advantages over the traditional Laemmli technique including: i) a longer shelf life of the pre-cast gels ranging from 8 months to 1 year; ii) a broad separation range of molecular weights from 1 to 400 kDa depending of the type of gel used; and iii) greater versatility (range of acrylamide percentage, the type of gel, and the ionic composition of the running buffer).
The procedure described in this video article utilizes the Bis-Tris discontinuous buffer system with 4-12% Bis-Tris gradient gels and MES running buffer, as an illustration of how to perform a western-blot using the Invitrogen NuPAGE electrophoresis system. In our laboratory, we have obtained good and reproducible results for various biochemical applications using this western-blotting method.

This technical article describes a standard western-blotting procedure using the commercially available NuPAGE electrophoresis Mini-Gel system from Invitrogen.

Western Blot utilizando el Invitrogen NuPage Novex Bis Tris MiniGels

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the ...

Investigación

Biología

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

Western Blot: Preparación de muestras para la detección

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of microbiology was transformed with the advent of Anton van Leeuwenhoek&#39;s microscope, which allowed scientists to visualize prokaryotes for the first time. The development of the polymerase chain reaction (PCR) is one of those innovations that changed the course of molecular science with its impact spanning countless subdisciplines in biology. The theoretical process was outlined by Keppe and coworkers in 1971; however, it was another 14 years until the complete PCR procedure was described and experimentally applied by Kary Mullis while at Cetus Corporation in 1985. Automation and refinement of this technique progressed with the introduction of a thermal stable DNA polymerase from the bacterium Thermus aquaticus, consequently the name Taq DNA polymerase.
PCR is a powerful amplification technique that can generate an ample supply of a specific segment of DNA (i.e., an amplicon) from only a small amount of starting material (i.e., DNA template or target sequence). While straightforward and generally trouble-free, there are pitfalls that complicate the reaction producing spurious results. When PCR fails it can lead to many non-specific DNA products of varying sizes that appear as a ladder or smear of bands on agarose gels. Sometimes no products form at all. Another potential problem occurs when mutations are unintentionally introduced in the amplicons, resulting in a heterogeneous population of PCR products. PCR failures can become frustrating unless patience and careful troubleshooting are employed to sort out and solve the problem(s). This protocol outlines the basic principles of PCR, provides a methodology that will result in amplification of most target sequences, and presents strategies for optimizing a reaction. By following this PCR guide, students should be able to: 
 
&#9679; Set up reactions and thermal cycling conditions for a conventional PCR experiment 
&#9679; Understand the function of various reaction components and their overall effect on a PCR experiment 
&#9679; Design and optimize a PCR experiment for any DNA template 
&#9679; Troubleshoot failed PCR experiments

PCR has emerged as a common technique in many molecular biology laboratories. Provided here is a quick guide to several conventional PCR protocols. Because each reaction is a unique experiment, optimal conditions required to generate a product vary. Understanding the variables in a reaction will greatly enhance troubleshooting efficiency, thereby increasing the chance to obtain the desired result.

Reacción en cadena: Protocolo Básico Plus Estrategias de resolución de problemas y optimización

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of ...

V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black box&#34; because an experimentalist does not know if it has been performed successfully until the last of several steps. Moreover, the quality of western blot data is sometimes challenged due to a lack of effective quality control tools in place throughout the western blotting process. Here we describe the V3 western workflow, which applies stain-free technology to address the major concerns associated with the traditional western blot protocol. This workflow allows researchers: 1) to run a gel in about 20-30 min; 2) to visualize sample separation quality within 5 min after the gel run; 3) to transfer proteins in 3-10 min; 4) to verify transfer efficiency quantitatively; and most importantly 5) to validate changes in the level of the protein of interest using total protein loading control. This novel approach eliminates the need of stripping and reprobing the blot for housekeeping proteins such as &#946;-actin, &#946;-tubulin, GAPDH, etc. The V3 stain-free workflow makes the western blot process faster, transparent, more quantitative&#160;and reliable.

V3 workflow is a western blot procedure using stain-free gels. The stain-free technology allows researchers to visualize protein separation quality, to verify the transfer efficiency, and most importantly, to validate the change in the protein of interest using total protein quantification as a reliable loading control.

Flujo de trabajo sin manchas V3 para un control de carga total de proteínas práctico, conveniente y confiable en Western Blotting

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black ...

A Practical Guide to Phylogenetics for Nonexperts

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this topic and so it presents inherent problems. Here we compile a practical introduction to phylogenetics for nonexperts.&#160;We outline in a step-by-step manner, a pipeline for generating reliable phylogenies from gene sequence datasets. We begin with a user-guide for similarity search tools via online interfaces as well as local executables. Next, we explore programs for generating multiple sequence alignments followed by protocols for using software to determine best-fit models of evolution. We then outline protocols for reconstructing phylogenetic relationships via maximum likelihood and Bayesian criteria&#160;and finally describe tools for visualizing phylogenetic trees. While this is not by any means an exhaustive description of phylogenetic approaches, it does provide the reader with practical starting information on key software applications commonly utilized by phylogeneticists. The vision for this article would be that it could serve as a practical training tool for researchers embarking on phylogenetic studies&#160;and also serve as an educational resource that could be incorporated into a classroom or teaching-lab.

Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis.&#160;

Una guía práctica para Phylogenetics para no expertos

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.

This protocol explores the latest advancements in performing Western blot analyses.&#160;These novel modifications employ a Bis-Tris gel system with a 35&#160;min electrophoresis run time, a 7&#160;min dry blotting transfer system, and infrared fluorescent protein detection and imaging that generates higher resolution, quality, sensitivity, and improved accuracy of Western data.

El más rápido de Occidente en la ciudad: un toque contemporáneo en el análisis clásico de Western Blot

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of ...

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.
In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.

Floral-Dip Transformation of Flax Linum usitatissimum to Generate Transgenic Progenies with a High Transformation Rate

Here, we present a protocol to transform flax using Agrobacterium-mediated plant transformation via floral-dip. This protocol is simple to perform and inexpensive, yet yields a higher transformation rate than the current available methods for flax transformation.

Floral-Dip transformación de lino (<em&gt; Linum usitatissimum</em&gt;) Para generar transgénicos progenies con una alta tasa de Transformación

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded &#180;indefinitely&#180;. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.

El código de barras genético con proteínas fluorescentes para aplicaciones multiplexados

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.

Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

Desarrollo de una polimerasa de amplificación ensayo cuantitativo recombinasa con un control positivo interno

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

La mucina en gel de agarosa de electroforesis: Western Blotting para glicoproteínas de alta peso molecular


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...