Este trabajo fue apoyado por la División de Investigación Intramuros, Instituto Nacional del Corazón, Pulmón y Sangre, Institutos Nacionales de Salud. S.H. fue apoyado por el Programa de Estudio estudiantil de asociación público-privada de Japón en el extranjero, y H.N. y K.Y. fueron por Valor y V Beca de Capacitación en El Extranjero de Drogas.

1. SDS-PAGE y transferir a una membrana PVDF
<ol><li>Realice SDS-PAGE para separar las proteínas en función del tamaño relativo. NOTA: Cualquier sistema de gel comercial o casero está bien. Por favor, siga el protocolo del fabricante.</li>
	<li>Transfiera proteínas separadas de un gel a una membrana PVDF. NOTA: Los métodos de transferencia comunes (transferencia semi-seca o húmeda) son adecuados. Por favor, siga el protocolo del fabricante.</li>
</ol>2. Bloqueo de membranas PVDF
<ol><li>Incubar las membranas en amortiguadores de bloqueo adecuados durante 1 h con agitación a temperatura ambiente. NOTA: Dependiendo del anticuerpo principal y del sistema de detección, se debe seleccionar un búfer de bloqueo adecuado. Por ejemplo, los bloqueadores más típicos son BSA, leche seca sin grasa, caseína y polímeros sintéticos comerciales. PBS y/o TBS con 0,1% Tween20 son los búferes más utilizados. Este paso de bloqueo se puede realizar durante la noche a 4 °C.</li>
</ol>3. Incubación con anticuerpo primario
<ol><li>Prepare una solución agente-1 que mejore la inmunorreacción al 10% (IRE-1) con agua destilada. Vórtice bien.</li>
	<li>Prepare el anticuerpo primario diluido con un 10% IRE-1. Mézclalo suave y a fondo.</li>
	<li>Si utiliza una membrana más grande (4 cm x 8 cm – 8 cm x 8 cm), agregue 8 ml de la solución de anticuerpos en tubos centrífugas cónicos de 50 ml. Si utiliza una membrana más pequeña (2 cm x 8 cm – 4 cm x 8 cm), agregue 3 ml de la solución de anticuerpos en tubo de polipropileno de fondo redondo de 14 ml (Tabla de materiales).</li>
	<li>Recoja la membrana PVDF con pinzas y escurra brevemente la solución de bloqueo. Inserte la membrana PVDF en este tubo con los dedos enguantados y asegúrese de que toda la membrana se adhiere a la pared del tubo. Cuando la membrana PVDF se inserta en un tubo, se enfrenta al "lado proteico" de la membrana que originalmente estaba en contacto con el gel hacia adentro. Cierre bien la tapa.</li>
	<li>Inserte el tubo de 50 ml en una botella de vidrio para el horno de hibridación. NOTA: Cuando se utilicen tubos de 14 ml para esta incubación, inserte un tubo de 14 ml en el tubo de 50 ml, utilizando un par de soportes de anillo de plástico para mantener el tubo interno en el centro del tubo de 50 ml.</li>
	<li>Encienda el horno de hibridación.</li>
	<li>Incubar la membrana con rotación de 6 rpm durante al menos 5 minutos. NOTA: Asegúrese de que la membrana se adhiere a la pared en todo momento y de que el tubo (interno) es horizontal para cubrir la membrana con la solución de anticuerpos uniformemente. Calibrar la dilución del anticuerpo y el tiempo de incubación antes del experimento real.</li>
</ol>4. Lavado de membrana
<ol><li>Detenga la rotación.</li>
	<li>Retire cuidadosamente la membrana PVDF del tubo con pinzas y coloque la membrana en un recipiente con 50 ml de PBS-T.</li>
	<li>Enjuague brevemente la membrana con PBS-T.</li>
	<li>Enjuague la membrana del recipiente con agua destilada hasta que no aparezcan burbujas. Esto es para eliminar la mayoría del anticuerpo.</li>
	<li>Transfiera la membrana PVDF del recipiente al hilandero de ensalada que contiene 250 ml de PBS-T.</li>
	<li>Coloque la cesta del colador en el spinner. Asegúrese de que la tapa se haya puesto de forma segura.</li>
	<li>Activa el spinner y corre durante unos 20-30 s.</li>
	<li>Deseche la solución y enjuague brevemente el interior del hilandero con agua destilada.</li>
	<li>Agregue 250 mL de PBS-T en el spinner otra vez.</li>
	<li>Repita los pasos 4.6 y 4.7.</li>
</ol>5. Incubación con el anticuerpo secundario
<ol><li>Prepare una solución agente-2 que mejore la inmunorreacción al 10% (IRE-2) con agua destilada. Vórtice bien.</li>
	<li>Prepare el anticuerpo secundario diluido con un 10% IRE-2. Mézclalo suave y a fondo.</li>
	<li>Seleccione el volumen de la solución de anticuerpos y el tubo como se indica en el paso 3.3.</li>
	<li>Recoja la membrana PVDF con pinzas y escurra la solución brevemente. Inserte la membrana PVDF en el tubo como se indica en el paso 3.4.</li>
	<li>Inserte el tubo de 50 ml en una botella de vidrio para el horno de hibridación.</li>
	<li>Encienda el horno de hibridación.</li>
	<li>Incubar la membrana con rotación de 6 rpm durante al menos 5 minutos. Asegúrese de que la membrana se adhiere a la pared en todo momento y de que el tubo (interno) es horizontal para cubrir uniformemente la membrana con la solución de anticuerpos. NOTA: Calibrar el tiempo de incubación antes del experimento real. Cuando el anticuerpo secundario esté conjugado fluorescentemente, realice la incubación en la oscuridad.</li>
</ol>6. Lavado de membrana
<ol><li>Siga los pasos 4.1 a 4.10.</li>
</ol>7. Adquisición de imágenes
<ol><li>Detección de quimioluminiscentes<ol>
<li>Coloque una pieza de película flexible semi-trasplantada (10 cm x 15 cm) sobre una superficie plana.</li>
		<li>Mezcle 2 componentes del sustrato quimioluminiscente en un tubo de 5 ml.</li>
		<li>Transfiera inmediatamente 1,5 ml del sustrato mixto a la película flexible semi-trasplante y coloque la membrana PVDF sobre ella. A continuación, transfiera el resto de la solución de sustrato a la membrana.</li>
		<li>Incubar la membrana PVDF con el sustrato mixto en una película flexible semitransparente durante 1 min.</li>
		<li>Recoja la membrana PVDF con pinzas y escurra brevemente la solución de sustrato.</li>
		<li>Empareje la membrana entre un par de películas de transparencia.</li>
		<li>Adquiera la imagen bajo modo quimioluminiscente.</li>
	</ol>
</li>
	<li>Detección fluorescente<ol>
<li>Recoja la membrana PVDF con pinzas y escurra la solución brevemente.</li>
		<li>Empareje la membrana entre un par de películas de transparencia.</li>
		<li>Adquiera la imagen en modo fluorescente. NOTA: Los pasos 7.1 y 7.2 deben realizarse cerca de la máquina de imágenes para garantizar la máxima sensibilidad.</li>
		<li>Cuando la señal de fondo es alta, realice un paso de lavado en un recipiente con 80-100 ml de PBS-T: 10 minutos de enjuague 3 veces para el anticuerpo primario y 5 minutos de enjuague 6 veces para el anticuerpo secundario. NOTA: Los anticuerpos primarios y secundarios diluidos con soluciones IRE del 10% se pueden utilizar hasta 8-10 veces sin pérdida de sensibilidad6. La solución debe mantenerse a 4 °C durante un mes.</li>
	</ol>
</li>
</ol>

<ol>
	<li>MacPhee, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+considerations+for+improving+Western+blot+analysis.">Methodological considerations for improving Western blot analysis.</a> Journal of Pharmacol Toxicology Methods. 61 (2), 171-177 (2010).</li><li>Janes, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+critical+factors+for+quantitative+immunoblotting.">An analysis of critical factors for quantitative immunoblotting.</a> Science Signaling. 8 (371), (2015).</li><li>Schuck, P., Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mass+transport+limitation+and+surface+heterogeneity+in+the+biophysical+characterization+of+macromolecular+binding+processes+by+SPR+biosensing.">The role of mass transport limitation and surface heterogeneity in the biophysical characterization of macromolecular binding processes by SPR biosensing.</a> Methods in Molecular Biology. 627, 15-54 (2010).</li><li>Li, J., Zrazhevskiy, P., Gao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eliminating+Size-Associated+Diffusion+Constraints+for+Rapid+On-Surface+Bioassays+with+Nanoparticle+Probes.">Eliminating Size-Associated Diffusion Constraints for Rapid On-Surface Bioassays with Nanoparticle Probes.</a> Small. 12 (8), 1035-1043 (2016).</li><li>Higashi, 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=Old+but+not+Obsolete:+An+Enhanced+High+Speed+Immunoblot.">Old but not Obsolete: An Enhanced High Speed Immunoblot.</a> Journal of Biochemistry. 168 (1), 15-22 (2020).</li><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> Proceedings of the National Academy of Sciences of the United States of America. 76, 4350-4354 (1979).</li><li>Burnette, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Western+blotting":+electrophoretic+transfer+of+proteins+from+sodium+dodecyl+sulfate--polyacrylamide+gels+to+unmodified+nitrocellulose+and+radiographic+detection+with+antibody+and+radioiodinated+protein+A.">"Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.</a> Analytical Biochemistry. 112 (2), 195-203 (1981).</li><li>Mishra, M., Tiwari, S., Gomes, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+purification+and+analysis:+next+generation+Western+blotting+techniques.">Protein purification and analysis: next generation Western blotting techniques.</a> Expert Review of Proteomics. 14 (11), 1037-1053 (2017).</li><li>Ciaccio, M. F., Wagner, J. P., Chuu, C. P., Lauffenburger, D. A., Jones, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems+analysis+of+EGF+receptor+signaling+dynamics+with+microwestern+arrays.">Systems analysis of EGF receptor signaling dynamics with microwestern arrays.</a> Nature Methods. 7 (2), 148-155 (2010).</li><li>Treindl, F., 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=A+bead-based+western+for+high-throughput+cellular+signal+transduction+analyses.">A bead-based western for high-throughput cellular signal transduction analyses.</a> Nature Communications. 7, 12852 (2016).</li><li>Sajjad, S., Do, M. T., Shin, H. S., Yoon, T. S., Kang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+western+blot+assay+by+rotational+cyclic+draining+and+replenishing+procedure.">Rapid and efficient western blot assay by rotational cyclic draining and replenishing procedure.</a> Electrophoresis. 39 (23), 2974-2978 (2018).</li><li>Kurien, B. T., Scofield, R. 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Los autores no han declarado intereses competidores directamente relevantes para el contenido de este artículo.

Western blot es una técnica analítica ampliamente utilizada para detectar proteínas específicas que se desarrolló ~Hace 40 años7,8. Desde entonces, la técnica ha seguido evolucionando, con innovaciones posteriores mejorando la sensibilidad, velocidad y cuantificación de la técnica2,9,10,11,12,...

Una mancha occidental (también conocida como inmunoblot) es una técnica poderosa y fundamental en una amplia gama de disciplinas científicas y clínicas. Esta técnica se utiliza para examinar la presencia, la abundancia relativa, la masa molecular relativa y las modificaciones post-traslacionales de las proteínas1. Combinado con el análisis de imágenes digitales, este método puede analizar de forma fiable la abundancia de proteínas y modificaciones de proteínas2. Aunque la mancha occidental se realiza de forma rutinaria, es un método que consume mucho tiempo y requiere mucho trabajo. Se requieren largas incubaciones del anticuerpo con membranas. Aquí, describimos una modificación del método de incubación que supera esta limitación sin sacrificar la sensibilidad.
Durante la incubación de la membrana, los anticuerpos flotan en solución mientras que los antígenos se inmovilizan en la membrana. Debido a su alta afinidad, la tasa de unión de anticuerpos al antígeno es más rápida que la difusión de anticuerpos de la solución a granel a la membrana. Esto crea una "capa de agotamiento" de baja concentración (Figura 1). Los anticuerpos más distantes pueden tardar horas en llegar a la membrana a través de la difusión pasiva, que es el principal factor responsable de los largos tiempos de incubación en esta técnica. Este efecto se denomina limitación de transporte masivo (MTL)3. Se ha propuesto que el drenaje repetitivo y la reposición de la solución que contiene anticuerpos puedan interrumpir la capa de agotamiento y superar el MTL4. Aquí, hemos ideado una técnica única que implementa este concepto cíclico de drenaje y reposición (CDR) para disminuir el efecto de MTL en un protocolo tradicional de hinchazón occidental y acortar notablemente el período de incubación requerido.
Con el fin de mantener la sensibilidad de detección con un corto tiempo de incubación, hicimos uso de una tecnología de mejora de la inmunorreacción que acelera la reacción antígeno-anticuerpo, mejorando así la relación señal-ruido5. Muchos agentes de mejora de inmunorreacción disponibles comercialmente (IRE) constan de 2 componentes (Solución 1 y 2, véase Tabla de Materiales). La composición o mecanismo de acción patentado de la IRE no se ha divulgado, pero hemos encontrado previamente que la IRE disminuyó la constante de disociación entre el antígeno y el anticuerpo en ensayos de unión de fase sólida, lo que indica que el aumento de la afinidad es, al menos en parte, responsable del efecto de mejora del IRE6. La combinación de CDR con IRE produce un protocolo de distensión occidental de ultra alta velocidad que reduce todo el tiempo de procedimiento sin sacrificar la sensibilidad.

<table><tbody><tr><td>14 mL Round Bottom High Clarity PP Test Tube, Graduated, with Snap Cap</td><td>Falcon</td><td>352059</td><td></td></tr><tr><td>Apollo Transparency Film for Laser Printers</td><td>N/A</td><td>N/A</td><td>Any kinds of Laser Printer Transparency Film are fine.</td></tr><tr><td>Azure Imaging System 600</td><td>Azure Biosystems</td><td>Azure 600</td><td>Any kind of Image Acquiring Sytem is fine for chemiluminescent and/or fluorescent detection.</td></tr><tr><td>Can Get Signal Immunoreaction Enhancer Solution</td><td>TOYOBO</td><td>NKB-101</td><td></td></tr><tr><td>CARNATION Instant Nonfat Dry Milk</td><td>Carnation</td><td>N/A</td><td></td></tr><tr><td>ECL anti-mouse IgG, Horseradish peroxidase linked F(ab&amp;rsquo;)2 fragment from sheep</td><td>GE Healthcare</td><td>NA9310V</td><td></td></tr><tr><td>ECL anti-rabbit IgG, Horseradish peroxidase linked F(ab&amp;rsquo;)2 fragment from donkey</td><td>GE Healthcare</td><td>NA9340V</td><td></td></tr><tr><td>HYBAID Micro-4</td><td>N/A</td><td>N/A</td><td>Any hybridization oven is fine as long as the tubes are rotated evenly at a horizontal position.</td></tr><tr><td>Immobilon-P PVDF Membrane, 0.45 &amp;micro;m pore size</td><td>Millipore Sigma</td><td>IPVH304F0</td><td></td></tr><tr><td>IRDye 680RD Goat anti-Mouse IgG Secondary Antibody</td><td>LICOR</td><td>926-68070</td><td></td></tr><tr><td>IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody</td><td>LICOR</td><td>926-32211</td><td></td></tr><tr><td>KPL LumiGLO Reserve Chemiluminescent Substrate</td><td>seracare</td><td>5430-0049</td><td></td></tr><tr><td>Mouse anti-&amp;szlig; actin antibody</td><td>Millipore Sigma</td><td>A5316</td><td></td></tr><tr><td>Odyssey Blocking Buffer (PBS)</td><td>LICOR</td><td>927-40100</td><td>Blocking Buffer for fluorescent detection</td></tr><tr><td>OXO Salad Spinner</td><td>OXO</td><td>32480V2B</td><td>Any salad spinner is fine as long as the PVDF membranes are rinsed vigorously without tear.</td></tr><tr><td>Parafilm Sealing Film</td><td>Bemis</td><td>Parafilm M PM996</td><td>semi-transparent flexible film</td></tr><tr><td>Polyethylene Flat-Top Screw Caps for 50 mL Conical Bottom Centrifuge Tubes</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Rings to hold 14 ml tube in the center of 50 ml tube</td><td>N/A</td><td>N/A</td><td>Prepared in a machine shop</td></tr><tr><td>Rabbit anti-6X His tag antibody</td><td>Abcam</td><td>ab9108</td><td></td></tr><tr><td>Tween20</td><td>Millipore Sigma</td><td>P2287</td><td></td></tr></tbody></table>

ultra-high-speed western blot using immunoreaction enhancing technology

Una mancha occidental (también conocida como inmunoblot) es un método canónico para la investigación biomédica. Se utiliza comúnmente para determinar el tamaño relativo y la abundancia de proteínas específicas, así como modificaciones de proteínas post-traslacionales. Esta técnica tiene una rica historia y permanece en uso generalizado debido a su simplicidad. Sin embargo, el procedimiento de hinchazón occidental famosamente toma horas, incluso días, para completar, con un cuello de botella crítico siendo los largos tiempos de incubación que limitan su rendimiento. Estos pasos de incubación son necesarios debido a la lenta difusión de anticuerpos de la solución a granel a los antígenos inmovilizados en la membrana: la concentración de anticuerpos cerca de la membrana es mucho menor que la concentración a granel. Aquí, presentamos una innovación que reduce drásticamente estos intervalos de incubación mediante la mejora de la unión de antígenos a través del drenaje cíclico y la reposición (CDR) de la solución de anticuerpos. También utilizamos una tecnología de mejora de la inmunorreacción para preservar la sensibilidad del ensayo. Una combinación del método CDR con un agente comercial de mejora de la inmunorreacción aumentó la señal de salida y redujo sustancialmente el tiempo de incubación de anticuerpos. La mancha occidental de ultra alta velocidad resultante se puede lograr en 20 minutos sin ninguna pérdida de sensibilidad. Este método se puede aplicar a las manchas occidentales utilizando detección quimioluminiscente y fluorescente. Este sencillo protocolo permite a los investigadores explorar mejor el análisis de la expresión proteica en muchas muestras.

Este ejemplo ilustra la eficacia del método CDR junto con la tecnología de inmunoenhancing en western blot. La incubación estática se realizó en una película flexible semitransparente donde las membranas PVDF fueron incubadas con la solución de anticuerpos, mientras que la incubación de CDR fue en un horno de hibridación mediante tubos giratorios que contenían las membranas y la solución de anticuerpos (Movie). La Figura 2 mostró cantidades de antígeno y anticuerpos, respectivam...

Watch this Scientific Journal Video about Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology at JoVE.com

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

Una técnica de distensión occidental de ultra alta velocidad se desarrolla mejorando la cinética de la unión antígeno-anticuerpo a través de la tecnología de drenaje cíclico y reposición (CDR) junto con un agente que mejora la inmunorreacción.

Blot occidental de ultra alta velocidad usando tecnología de mejora de la inmunorreacción

A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and ...

ultra-high-speed-western-blot-using-immunoreaction-enhancing

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

5.8K vistas.  National Institutes of Health. Una técnica de distensión occidental de ultra alta velocidad se desarrolla mejorando la cinética de la unión antígeno-anticuerpo a través de la tecnología de drenaje cíclico y reposición (CDR) junto con un agente que mejora la inmunorreacción.

Video: Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

Un protocolo optimizado para el ensayo de cambio de movilidad electroforética utilizando oligonucleótidos marcados con colorante fluorescente infrarrojo

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

Investigación

Bioquímica

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 ...

Biología

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

Detección altamente sensible y cuantitativa de las proteínas y sus isoformas por método capilar isoeléctrica

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides ...

Immunoblot Analysis

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. It involves the solubilization and electrophoretic separation of proteins, glycoproteins, or lipopolysaccharides by gel electrophoresis, followed by quantitative transfer and irreversible binding to nitrocellulose, PVDF, or nylon. The immunoblotting technique has been useful in identifying specific antigens recognized by polyclonal or monoclonal antibodies and is highly sensitive (1 ng of antigen can be detected). This unit provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. This video provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

El análisis por inmunotransferencia

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the ...

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 ...

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 ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

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 within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete 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 ...

Immunoprecipitation-Based Techniques: Purification of Endogenous Proteins Using Agarose Beads

Source: Susannah C. Shissler1, Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Immunoprecipitation (IP, also known as a &#39;pull-down&#39; assay) is a widely used technique that has applications in a variety of fields. First conceived in 1984, it was refined in 1988 (1, 2). The fundamental goal of IP is purification and isolation of a specific protein using an antibody against that protein. The word &#34;immuno&#34; refers to the use of an antibody while the word &#34;precipitation&#34; refers to pulling down a specific substance from a solution. The target protein might be endogenous or recombinant. Most recombinant proteins have an epitope tag (i.e. myc or flag) attached to them to simplify subsequent purification. Typically, it is easier to optimize recombinant protein IP because the antibodies against recombinant epitope tags are very strong and effective. Antibodies against endogenous proteins have extremely variable efficacy - making it much more difficult to optimize these IPs. A necessary step after immunoprecipitation is verification of purification. The isolated protein is resolved using SDS-PAGE and subsequently probed for purity by western blots (Figure 1). An important control is the use of a different antibody during the Western blot to verify pull down of the correct protein. The combination of IP with subsequent techniques is a powerful analysis tool. The goal after purification may be characterization of the protein itself by NMR, mass spectrometry, and in vitro assays, or analysis of the protein&#39;s interacting partners (i.e. protein, DNA, RNA) (3, 4, 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10502/10502fig1.jpg" /> 
Figure 1: Overview of Immunoprecipitation Procedure. Immunoprecipitation is the isolation of a specific protein using an antibody. After production of lysate from cells, there are two major steps- pre-clearing and pull down. During pre-clearing step, the cell-lysates are pre-cleared of proteins that bind to antibodies non-specifically using an isotype control antibody. In pull down step, the target protein is pulled down using a protein-specific antibody. The isolated protein is then analyzed by Western blot. Isotype antibodies and protein specific antibodies have the same constant domain, but different antigen binding domains. A key component of this protocol is Protein A/G agarose beads that bind the constant domain of antibodies- allowing immunoprecipitation of the target protein. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/10502/10502fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Antibodies are the key component of an immunoprecipitation that differentiate it from other forms of protein purification (i.e. nickel affinity column purification). Antibodies are molecules made by B cells that can recognize specific protein epitopes. Antibodies have two domains: constant (Fc) and antigen binding (Fab) (Figure 1). The constant domain identifies the type of antibody and dictates function in vivo. Usually, the constant domains of antibodies used for IP are mouse, rat, or rabbit IgG. The antigen binding portion of the antibody recognizes a specific epitope of a specific protein. Antibodies can recognize epitopes on folded proteins that may not exist when the protein is denatured and vice versa. Therefore, the availability of the epitope depends on protein folding - identifying an important factor to consider when choosing antibodies and conditions for IP.
Both prokaryotic and eukaryotic systems have antibody-binding proteins. In eukaryotic systems, the purpose is immune protection from bacteria while in prokaryotic systems, the purpose is protection from the immune system. Antibody-binding proteins affect IP methodology in two ways. First, there is a necessary pre-clearing step (Figure 1) to rid the lysate of proteins that bind antibodies - thereby reducing non-specific binding in the final product. This step uses an isotype antibody that has the same constant domain as but a different antibody binding domain than your protein-specific antibody. Bacterial antibody-binding proteins are the second key component of this method. After the protein-specific antibody binds the target protein, the antibody: protein complex must be pulled down (Figure 1). Proteins A, G, and L are bacterial proteins that bind the constant domain of antibodies. While bacteria use this to subvert the immune system, researchers have co-opted this system for easy antibody purification, and it is used during both the pre-clearing and pull-down steps. These proteins have different binding affinities for different species and different constant domain subtypes - another factor to consider when choosing conditions for IP. Many companies sell Protein A/G labeled agarose beads (Figure 1), pre-made spin columns, or resins to make columns. In general, beads and spin columns are used for smaller sample sizes while resins are used for bulk purification.
In this lab exercise, we demonstrate how to purify the endogenous protein c-myc, from primary murine thymocytes, using Protein A/G Plus agarose beads based basic immunoprecipitation technique. The protocol starts from cell lysate preparation and ends with the with verification of successful protein pull down using Western blot analysis.

Immunoprecipitation Techniques: Purification of Proteins

Técnicas basadas en inmunoprecipitación: purificación de proteínas endógenas mediante perlas de agarosa

Source: Susannah C. Shissler1, Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Immunoprecipitation ...

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Inmunología

The Western Blot

Western Blotting is used to identify the presence of specific proteins in electrophoretically separated samples. Following separation by a technique known as sodium dodecyl sulfate polyacrylamide gel electrophoresis, or SDS-PAGE, western transfer is used to move proteins from a polyacrylamide gel onto a piece of membrane which traps the proteins in their respective locations. Next, the membranes are probed with antibodies in a process called immunboblotting. Immunoblotting uses antibody-protein and antibody-antibody binding through specific recognition sites, providing the high specificity required for identifying a single protein. The detection of antibodies takes place using reporter systems which includes the use of enzymes. Enzymes can be attached to the end of an antibody and react with substrates to produce changes in color or light. These signals can then be imaged and quantified using a process called densitometry.
This video-article presents an overview of the western blot technique by describing western transfer, the use of antibody detection, and image analysis. The steps involved with western transfer such as the assembly of the transfer sandwich and transfer conditions are discussed in detail as well as the theory behind antibody binding and detection of those antibodies. The broad applications of this technique are described through several examples including the detection of protein-protein interactions and identification of individual proteins within protein complexes.

Western Blotting: Western Transfer, Antibody Detection, and Image Analysis

Western Blot

Western Blotting is used to identify the presence of specific proteins in electrophoretically separated samples.  Following separation by a technique ...

Biología básica


Métodos Básicos en Biología Celular y Molecular

Chemiluminescent Western Blot Assay for Protein Processing

Source: Hillert-Richter, L. K. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/62842">Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex.</a> J. Vis. Exp. (2021).
This video describes the chemiluminescence-based western blotting of immunoprecipitated lysates. The technique helps determine protein processing and activation. The chemiluminescent signal detected is proportional to the level of protein processing during its activation.

Ensayo de Western Blot quimioluminiscente para el procesamiento de proteínas

Source: Hillert-Richter, L. K. et al., Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex. J. ...