Este trabajo fue apoyado por el consejo de investigación académica de Singapur (ARC) [número de concesión 90/07]. Damos las gracias por el apoyo radiodurans.

El protocolo de texto completo y detallado de este procedimiento experimental está disponible en los protocolos actuales en biología molecular. Detalladas paso a paso las instrucciones para el montaje del bocadillo de gel y gel para los aparatos se pueden encontrar en el sitio web BioRad 3. Equipo necesario: Placas de vidrio (interior y exterior) 10 cm de células: 10,1 x 7,3 cm (placa interior), 10,1 x 8,2 cm (la placa exterior), BioRad 20 cm de células: 20 x 20 cm (placa interior), 20 x 22,3 cm (placa exterior), BioRad 0.5-1.5 mm peine de gel y espaciadores Gel de pie de fundición Gel aparato con tapa y cables Suministro de alta tensión El bloque de calentamiento o baño de agua Pipetas serológicas y la ayuda de pipetas Pipeta y puntas de pipeta Gel secador o un escáner Reactivos y soluciones: Urea (ultrapura) 40% de poliacrilamida solución (29:1) 10 x TBE solución (Tris-Borato, EDTA) Agua desionizada, destilada TEMED 30% (w / v) de persulfato de amonio 0,5 x TBE solución Formamida EDTA Cyanol xileno Azul de bromofenol Metanol Etanol <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Volumen </td><td colspan="3" style="text-align:center;"> 50 ml </td><td colspan="3" style="text-align:center;"> 60 ml </td><td colspan="3" style="text-align:center;"> 10 ml </td></tr><tr><td> Acrilamida concentración </td><td> 10% </td><td> 12,5% </td><td> 15% </td><td> 10% </td><td> 12,5% </td><td> 15% </td><td> 10% </td><td> 12,5% </td><td> 15% </td></tr><tr><td> g de urea </td><td> 24 </td><td> 24 </td><td> 24 </td><td> 28.8 </td><td> 28.8 </td><td> 28.8 </td><td> 4.8 </td><td> 4.8 </td><td> 4.8 </td></tr><tr><td> ml 40% acrílico (29:1) </td><td> 12.5 </td><td> 15.625 </td><td> 18.75 </td><td> 15 </td><td> 18.75 </td><td> 22.5 </td><td> 2.5 </td><td> 3.125 </td><td> 3.75 </td></tr><tr><td> UL 30% de APS </td><td> 166 </td><td> 166 </td><td> 166 </td><td> 199.2 </td><td> 199.2 </td><td> 199.2 </td><td> 33 </td><td> 33 </td><td> 33 </td></tr><tr><td> ul TEMED </td><td> 20 </td><td> 20 </td><td> 20 </td><td> 24 </td><td> 24 </td><td> 24 </td><td> 4 </td><td> 4 </td><td> 4 </td></tr><tr><td> 10 x TBE </td><td> 5 </td><td> 5 </td><td> 5 </td><td> 6 </td><td> 6 </td><td> 6 </td><td> 1 </td><td> 1 </td><td> 1 </td></tr><tr><td colspan="10"> Llenar el volumen con agua desionizada, destilada </td></tr></tbody></table> Tabla 1: Reactivos y soluciones necesarias para los diferentes concentraciones de acrilamida y los volúmenes para la resolución de un solo varados oligonucleótidos Gel de montaje sandwich y gel de preparación <ol><li> Montar el gel de acuerdo a la descripción de los fabricantes y fijar el gel en la cámara de gel-casting 3. Utilice separadores de 0.5-1.5 mm de espesor. </li><li> Prepare la solución de poliacrilamida apropiada de acuerdo con los protocolos actuales en biología molecular o como se indica en la Tabla 1. Para un gel de acrilamida desnaturalizante de 20 cm x 22 cm x 1,5 mm, 60 ml de solución de gel y un 10,1 x 8,2 cm x 1 mm de gel de solución al 5 ml de gel es suficiente. Mayor geles se utilizan cuando los productos esperados / las bandas están dentro del rango de unas pocas bases, a continuación, un gel ya se resolverán las bandas con una diferencia de un solo nucleótido. Si las bandas se espera difieren en más de 10 nucleótidos, más pequeños geles se permite la resolución de los fragmentos. Elegir el porcentaje de poliacrilamida que se adapte a sus necesidades de separación, una mayor concentración de poliacrilamida resolverá menor fragmentos de peso molecular. El uso de urea ultrapura y se mezclan con la cantidad deseada de la acrilamida. Añadir tampón TBE a la mezcla de gel para conseguir una concentración final de 0,5 a 1 x TBE y llenar el volumen con agua desionizada, destilada. Se calienta la solución durante 20 segundos en el microondas y mezclar suavemente. Para grandes volúmenes de gel repetir este paso hasta que la solución es la mano caliente. </li><li> Verter el gel inmediatamente con una pipeta serológica y una pipeta automática de la ayuda entre las dos placas de vidrio. Evitar la introducción de burbujas de aire. Inserte el peine y dejar polimerizar el gel durante 30-60 minutos. </li></ol> La instalación del equipo de electroforesis y el gel prerun <ol><li> Desmontar el gel de la cámara de fundición y montaje para el aparato de gel de acuerdo con las instrucciones del fabricante 3. </li><li> Llenar la cámara baja del buffer ingenio en funcionamiento (0,5-1 x TBE) que las placas de vidrio se subse fusionó con 2-3 cm de amortiguamiento. Llenar la cámara de amortiguación superior hasta la parte superior del gel con el funcionamiento de amortiguación. </li><li> Retire con cuidado el peine y lave los pocillos con tampón en ejecución mediante una pipeta y puntas de carga de gel. </li><li> Fijar la tapa del sistema de gel y conectar los cables a una fuente de alimentación de alta tensión. Antes de poder cargar las muestras que tiene que prerun el gel por lo menos 30 minutos para calentar el gel y para eliminar la urea restante del gel. La temperatura óptima debe estar entre 45-55 ° C. Evite las temperaturas superiores a 60 ° C en forma de bandas podría manchar o las placas de vidrio se puede romper. Elija vatios constante para el prerun (15-25 W por gel). </li></ol> Preparación de la muestra <ol><li> Mientras tanto preparar las muestras. Por lo tanto, añadir la cantidad apropiada de la mezcla de carga 2 x gel de la muestra. La mezcla de carga por lo general contiene 90% formamida, EDTA 0,5%, 0,1% cyanol xileno y 0,1% azul de bromofenol. </li><li> Antes de que las muestras pueden ser cargados en el gel, las muestras deben ser el calor desnaturalizado por calentamiento de las muestras entre los 70-90 º C durante unos minutos. </li></ol> Cargar y ejecutar el gel <ol><li> Cuando el prerun haya terminado, retire la tapa y enjuagar los bolsillos bien como se describió anteriormente como la urea ha infiltrado en los pozos. </li><li> Aplicar las muestras cuidadosamente desde el fondo de la bolsa. Evitar la introducción de burbujas de aire. Tampón de carga se debe aplicar a los bolsillos vacíos para mantener la igualdad de condiciones durante la carrera. </li><li> Montar la tapa y ejecutar el gel en vatios constante para mantener una temperatura de gelificación de 55 ° C similar a la prerun. Observar la migración de los colorantes marcador hasta que el frente tinte alcanzado el extremo inferior del gel. La duración de ejecución depende del porcentaje de acrilamida usada, la fuerza iónica del buffer y grosor del gel. Una carrera puede durar entre 2-4 horas. Comprobar la temperatura del gel. Un termómetro de gel pueden ayudar a controlar la temperatura adecuada durante la carrera. </li></ol> Proceso del gel <ol><li> Cuando el frente de tinte ha llegado al final del gel se pone el gel de la cámara de amortiguación inferior. Quitar el gel de la cámara y aflojar las abrazaderas. Extraiga los separadores y cuidadosamente disimular las placas de vidrio. Si es necesario cortar la parte superior y que contiene parte del gel. </li><li> Transferir cuidadosamente el gel en un plato con una solución de gel de fijación (la misma concentración de TBE más metanol y el etanol 10.5%). Deje que el gel en la solución durante 5-10 minutos y cambiar el búfer dos veces. </li><li> El gel está listo para su posterior procesamiento con un secador de gel de vacío o de exploración directa del gel. </li></ol>

<ol>
	<li>Maniatis, T., Jeffrey, A., van deSande, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chain+length+determination+of+small+double-+and+single-stranded+DNA+molecules+by+polyacrylamide+gel+electrophoresis.">Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis.</a> Biochemistry. 14, 3787-3794 (1975).</li><li>Albright, L. M., Slatko, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Denaturing+polyacrylamide+gel+electrophoresis.">Denaturing polyacrylamide gel electrophoresis.</a> Curr Protoc Nucleic Acid Chem. , (2001).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PROTEAN II xi Cell and PROTEAN II xi 2-D Cell Instruction Manual. , (2001).</li></ol>

The authors have nothing to disclose.

<ol><li> La nitidez de la banda depende de varios factores. El volumen de muestra cargada de bandas de fuerte debe ser lo más pequeña posible, es decir, lo ideal es entre 1.5 l. Sin embargo, hasta 15 l puede ser cargado que todavía asegura una resolución aceptable. Menos de resultados de la muestra de volumen en las bandas más nítida. </li><li> La calidad de la banda también depende del grosor del gel. Diluyente geles, como por ejemplo 0,75 mm muestran mejores resultados que los geles 1,5 mm de espesor. </li><li> La forma de las bandas t...

denaturing urea polyacrylamide gel electrophoresis (urea page)

PÁGINA urea o desnaturalización de poliacrilamida electroforesis en gel de urea cuenta con 8.6 M de urea, lo que desnaturaliza el ADN o ARN estructuras secundarias y se utiliza para su separación en una matriz de gel de poliacrilamida en base al peso molecular. Fragmentos de entre 2 y 500 bases, con las diferencias de longitud tan pequeño como un solo nucleótido, pueden ser separados utilizando este método 1. La migración de la muestra depende de la concentración de acrilamida elegido. Un mayor porcentaje de poliacrilamida se resuelve fragmentos de menor peso molecular. La combinación de urea y temperaturas de 45-55 ° C durante la ejecución del gel permite la separación de ADN no estructurados o moléculas de ARN. En general, este método es necesario para analizar o purificar el ADN de cadena sencilla o fragmentos de ARN, tales como los oligonucleótidos sintetizados o etiqueta o de los productos de las reacciones de escisión enzimática. En este artículo de vídeo que muestran cómo preparar y ejecutar los geles de poliacrilamida desnaturalizantes urea. Consejos técnicos se incluyen, además de los 1,2 del protocolo original.

Watch this Scientific Journal Video about Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE at JoVE.com

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Poliacrilamida electroforesis en gel de urea se utiliza para separar una sola cadena de ADN o ARN, hasta un límite de 500 nucleótidos. Urea en combinación con el calor desnaturaliza las muestras y no estructurados cadenas simples migrar dentro de la matriz de gel de acuerdo a su peso molecular.

Poliacrilamida electroforesis en gel de urea (urea PÁGINA)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their ...

denaturing-urea-polyacrylamide-gel-electrophoresis-urea-page

Research

JoVE Journal

Biology

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

78.3K vistas.  Nanyang Technological University, Singapore - NTU. Poliacrilamida electroforesis en gel de urea se utiliza para separar una sola cadena de ADN o ARN, hasta un límite de 500 nucleótidos. Urea en combinación con el calor desnaturaliza las muestras y no estructurados cadenas simples migrar dentro de la matriz de gel de acuerdo a su peso molecular.

Video: Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis DGGE

Gradiente desnaturalizante electroforesis en gel (DGGE)

Investigación

Biología

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

La detección de la agregación amiloide de Semi-desnaturalización electroforesis en gel de agarosa-detergente

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches.
Several improvements of the original Coomassie protocol1 have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution2, and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol3. Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol4. The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins.
Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.

This video shall popularize a colloidal Coomassie G-250 staining protocol according to Kang et al. for the detection of average 4 ng protein in gels. The staining is completed within 2 hours and without any effort. We routinely use Kang's protocol for analytical purposes in gel-based proteomics.

Rápido y sensible coloidal Coomassie G-250 La tinción de proteínas en geles de poliacrilamida

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

Azul nativos electroforesis en gel de poliacrilamida (BN-PAGE) para el análisis de los complejos Multiproteinas de lisados ​​celulares

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel&#39;s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is &#34;biased reptation&#34;, whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 
 
1. Understand the mechanism by which DNA fragments are separated within a gel matrix 
2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 
3. Identify an agarose solution of appropriate concentration for their needs 
4. Prepare an agarose gel for electrophoresis of DNA samples 
5. Set up the gel electrophoresis apparatus and power supply 
6. Select an appropriate voltage for the separation of DNA fragments 
7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 
8. Determine the sizes of separated DNA fragments 
&#160;

A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

Electroforesis en gel de agarosa para la separación de los fragmentos de ADN

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.

Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.

Preparación de ADN-poliacrilamida reticulada hidrogeles

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

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 ...

Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb the assembly and stability of OXPHOS complexes. This protocol describes the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) in application to cultured human cells. First, mitochondria are extracted from the cells using digitonin, then using lauryl maltoside, the intact OXPHOS complexes are isolated from the mitochondrial membranes. The OXPHOS complexes are then resolved by gradient gel electrophoresis in the presence of the negatively charged dye, Coomassie blue, which prevents protein aggregation and ensures electrophoretic mobility of protein complexes towards the cathode. Finally, the OXPHOS complexes are detected by standard immunoblotting. Thus, BN-PAGE is a convenient and inexpensive technique that can be used to evaluate the assembly of entire OXPHOS complexes, in contrast to the basic SDS-PAGE allowing the study of only individual OXPHOS complex subunits.

This protocol demonstrates the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis. Here the method applied to cultured human cells is described.

Análisis de complejos de cadena respiratoria mitocondrial en células humanas cultivadas usando electroforesis en Gel de poliacrilamida nativo azul e Immunoblotting

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb ...

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role in a variety of basic biological structures and processes. As polymers, GAGs exist as a polydisperse mixture containing polysaccharide chains that can range from 4000 Da to well over 40,000 Da. Within these chains exists domains of sulfation, conferring a pattern of negative charge that facilitates interaction with positively charged residues of cognate protein ligands. Sulfated domains of GAGs must be of sufficient length to allow for these electrostatic interactions. To understand the function of GAGs in biological tissues, the investigator must be able to isolate, purify, and measure the size of GAGs. This report describes a practical and versatile polyacrylamide gel electrophoresis-based technique that can be leveraged to resolve relatively small differences in size between GAGs isolated from a variety of biological tissue types.

This report describes techniques to isolate and purify sulfated glycosaminoglycans (GAGs) from biological samples and a polyacrylamide gel electrophoresis approach to approximate their size. GAGs contribute to tissue structure and influence signaling processes via electrostatic interaction with proteins. GAG polymer length contributes to their binding affinity for cognate ligands.

Detección de glicosaminoglicanos por electroforesis en gel de poliacrilamida y tinción de plata

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role ...

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent adult integument. Their immobility and transparency make them ideal for in vivo live imaging experiments. Many studies have focused on the dorsal epithelial monolayer of the pupal&#160;notum because of its accessibility and relatively large size. In addition to the studies of epithelial mechanics and development, the notum has been an ideal tissue to study wound healing. After an injury, the entire epithelial repair process can be captured by live imaging over 6-12 h. Despite the popularity of the notum for live imaging, very few published studies have utilized fixed notum samples. Fixation and staining are common approaches for nearly all other Drosophila tissues, taking advantage of the large repertoire of simple cellular stains and antibodies. However, the pupal notum is fragile and prone to curling and distortion after removal from the body, making it challenging to complement live imaging. This protocol offers a straightforward method for fixing and staining the pupal notum, both intact and after laser-wounding. With this technique, the ventral side of the pupa is glued down to a coverslip to immobilize the pupa, and the notum is carefully removed, fixed, and stained. The notum epithelium is mounted on a slide or between two coverslips to facilitate imaging from the tissue&#39;s dorsal or ventral side.

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The present protocol details the preparation and visualization of fixed tissue of the Drosophila pupal notum. It can be used for either intact or wounded tissue, and the original architecture of the tissue is preserved. The procedures for dissecting, fixing, and staining are all described in this article.

Disección, fijación y visualización del Drosophila Pupal Notum

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent ...