Damos las gracias a Simon Alberti para obtener ayuda con el desarrollo de este protocolo. Este trabajo fue apoyado por una beca de los Institutos Nacionales de Salud (GM25874), Médico Howard Hughes Investigatorship Instituto (de SL), y una beca de la Fundación Nacional de Ciencia formación predoctoral (con HR).

<html> Parte 1: Preparación del gel <ol><li> Montar la bandeja de fundición de gel. El equipo estándar para la electroforesis de ADN horizontal se puede utilizar. Para un gran número de muestras, nos preparamos a 20 cm x 24 cm losa con un máximo de cuatro de 50 y peines. Asegúrese de que la losa no tiene arañazos, ya que pueden distorsionar la imagen blot. </li><li> Crear una solución de agarosa 1,5% (medio o alto gel resistencia y bajo EEO) en 1X TAE. El volumen debe ser suficiente para sumergir completamente los dientes del peine - que se desea cargar de la muestra tanto como sea posible para maximizar la detección. Microondas la mezcla hasta que la agarosa se disuelva por completo. </li><li> Suma rápida SDS al 0,1% de una reserva de 10%. Agitar para mezclar. Si algunos de agarosa solidifica durante este paso, disolver con un plato caliente y tener cuidado para evitar una ebullición. </li><li> Vierta la solución en la bandeja de fundición. Utilice un peine de rastrillo de las burbujas, ya que más tarde podrían interferir con la transferencia. </li><li> Después de gel, quite los peines y colocar el gel en el depósito de gel. Sumerja completamente el gel en 1X TAE conteniendo 0,1% SDS. </li></ol> Parte 2: Preparación de muestras <ol><li> De alto rendimiento para el análisis de los lisados ​​de levadura, se inicia con las culturas de 2 ml crecido durante la noche con agitación rápida en 96 pozos bloques. En este caso, cada cultura es una sobreexpresión de la proteína de interés. Al analizar las proteínas de baja abundancia, grandes cantidades de cultivos debe ser utilizado </li><li> Las células de la cosecha por centrifugación a 2000 rcf durante 5 min a temperatura ambiente. </li><li> Resuspender las células en el agua y se centrifuga de nuevo. </li><li> Resuspender en 1 ml de solución spheroplasting. Se incuba durante aproximadamente 30 minutos a 30 ° C (se puede confirmar la eficiencia spheroplasting por microscopía). </li><li> Centrifugar a 800 RCF durante 5 min a temperatura ambiente. Eliminar completamente el sobrenadante. </li><li> Resuspender esferoplastos pellets en 100 ml de solución amortiguadora de lisis. </li><li> Cubrir el bloque con la cinta y el vórtice de alta velocidad durante 2 min. </li><li> Precipitado celular escombros en 4000 rcf durante 2 min. </li><li> Retire con cuidado el sobrenadante a un nuevo contenedor, por ejemplo, una placa de 96 pocillos PCR. </li><li> Si lo desea, determinar la concentración de proteínas de los lisados. </li><li> Añadir la muestra de amortiguación 4X a las muestras para generar lisados ​​que contienen 1X tampón de la muestra. Incubar durante 5 minutos a temperatura ambiente. </li><li> Carga de gel. Si lo desea, guarde la mitad del volumen de la muestra y hervir durante análisis SDS-PAGE. Con el fin de monitorear el grado de transferencia más adelante, incluye un pre-SDS-PAGE teñido marcador. Además, un marcador de peso molecular que consiste en proteínas de gran tamaño (por ejemplo, pollo pectoral extracto) se puede utilizar para calcular los tamaños de los complejos de resolver. </li><li> Funcionar a baja tensión (≤ 3 V / cm la longitud del gel) hasta que el frente tinte alcance aproximadamente 1 cm del extremo del gel. Esto puede durar varias horas. Es importante que el gel de mantener la calma, de lo contrario la difusión de proteínas de bajo peso molecular (por ejemplo, los monómeros) puede limitar su detección. </li></ol> Parte 3: Transferencia <ol><li> Corte un pedazo de nitrocelulosa de las mismas dimensiones que el gel. </li><li> Cortar 20 piezas de GB004 y GB002 8 piezas de papel secante, con las mismas dimensiones que el gel. Cortar una pieza adicional de GB004 para ser utilizado como una mecha, que sea de unos 20 centímetros más ancho que el gel. </li><li> Sumergir las tiras de nitrocelulosa, mecha, y 4 piezas de GB002 en 1X TBS. </li><li> En un recipiente de plástico, reunir a un montón de papeles de la siguiente manera: 20 piezas de seco GB004, a continuación, cuatro piezas de GB002 seco, luego un trozo de pre-mojado GB002. Coloque las tiras de nitrocelulosa en la parte superior de la pila. </li><li> Enjuague el gel en la bandeja de fundición brevemente en agua para eliminar el exceso de tampón en funcionamiento. Entonces, cuidadosamente comenzar a deslizar el gel de la bandeja en la pila. Mientras desliza el gel de la bandeja, apagar la membrana con TBS según sea necesario. El buffer adicional ayuda a prevenir burbujas de quedar atrapados en el gel. Una pipeta de transferencia que funciona bien para este propósito. </li><li> Después de que el gel ha sido movido a la pila, revise si hay burbujas. Si están presentes, levantar el borde del gel y volver a aplicar buffer hasta que las burbujas pueden ser resueltos. </li><li> Puesto que los tres restantes pre-humedecida GB002 piezas en la parte superior del gel. Asegurar el contacto completo entre todas las capas de rodadura de una pipeta con firmeza en la parte superior de la pila. </li><li> El flanco de la pila de transferencia con dos bandejas que contienen elevados TBS. Coloque la mecha que ya húmedo en toda la pila de tal manera que cada extremo de la mecha se sumerge en TBS. </li><li> Cubra la pila de la transferencia de ensamblado con una bandeja de plástico adicionales que soportan el peso extra (por ejemplo, una botella de 500 ml de agua). </li><li> Permitir la transferencia de proceder por un mínimo de tres horas, o toda la noche. </li><li> Después de la transferencia, la membrana puede ser procesado por la norma de Western Blot. </li></ol> Solución Spheroplasting 1,2 M D-sorbitol 0,5 mM de MgCl 2 20 mM Tris, pH 7,5 50 mM BME (añadir nuevo) 0,5 mg / ml Zymolyase 100T (añadir nuevo) coration: underline; &quot;&gt; Tampón de lisis 100 mM Tris 7.5 50 mM NaCl 10 mM BME (añadir nuevo) inhibidores de la proteasa (añadir nuevo) Muestra de amortiguación 4X 2X TAE 20% de glicerol 8% SDS azul de bromofenol a la preferencia

<ol>
	<li>Kryndushkin, D. S., Alexandrov, I. M., Ter-Avanesyan, M. D., Kushnirov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+[PSI+]+prion+aggregates+are+formed+by+small+Sup35+polymers+fragmented+by+Hsp104.">Yeast [PSI+] prion aggregates are formed by small Sup35 polymers fragmented by Hsp104.</a> J. Biol. Chem. 278, 49636-49643 (2003).</li><li>Alexandrov, I. M., Vishnevskaya, A. B., Ter-Avanesyan, M. D., Kushnirov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appearance+and+Propagation+of+Polyglutamine-based+Amyloids+in+Yeast:+TYROSINE+RESIDUES+ENABLE+POLYMER+FRAGMENTATION.">Appearance and Propagation of Polyglutamine-based Amyloids in Yeast: TYROSINE RESIDUES ENABLE POLYMER FRAGMENTATION.</a> J. Biol. Chem. 283, 15185-15192 (2008).</li><li>Allen, K. 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=Hsp70+chaperones+as+modulators+of+prion+life+cycle:+novel+effects+of+Ssa+and+Ssb+on+the+Saccharomyces+cerevisiae+prion+[PSI+].">Hsp70 chaperones as modulators of prion life cycle: novel effects of Ssa and Ssb on the Saccharomyces cerevisiae prion [PSI+].</a> Genetics. 169, 1227-1242 (2005).</li><li>Aron, R., Higurashi, T., Sahi, C., Craig, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=J-protein+co-chaperone+Sis1+required+for+generation+of+[RNQ+]+seeds+necessary+for+prion+propagation..">J-protein co-chaperone Sis1 required for generation of [RNQ+] seeds necessary for prion propagation..</a> The EMBO journal. 26, 3794-3803 (2007).</li><li>Bagriantsev, S. N., Kushnirov, V. V., Liebman, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+amyloid+aggregates+using+agarose+gel+electrophoresis.">Analysis of amyloid aggregates using agarose gel electrophoresis.</a> Methods in Enzymology. 412, 33-48 (2006).</li><li>Borchsenius, A. S., Muller, S., Newnam, G. P., Inge-Vechtomov, S. G., Chernoff, Y. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+variant+maintained+only+at+high+levels+of+the+Hsp104+disaggregase.">Prion variant maintained only at high levels of the Hsp104 disaggregase.</a> Current Genetics. 49, 21-29 (2006).</li><li>Salnikova, A. B., Kryndushkin, D. S., Smirnov, V. N., Kushnirov, V. V., Ter-Avanesyan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonsense+suppression+in+yeast+cells+overproducing+Sup35+(eRF3)+is+caused+by+its+non-heritable+amyloids.">Nonsense suppression in yeast cells overproducing Sup35 (eRF3) is caused by its non-heritable amyloids.</a> J. Biol. Chem. 280, 8808-8812 (2005).</li><li>Tank, E. M., Harris, D. A., Desai, A. A., True, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+protein+repeat+expansion+results+in+increased+aggregation+and+reveals+phenotypic+variability.">Prion protein repeat expansion results in increased aggregation and reveals phenotypic variability.</a> Mol. Cell. Biol. 27, 5445-5455 (2007).</li><li>Douglas, P. 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=Chaperone-dependent+amyloid+assembly+protects+cells+from+prion+toxicity.">Chaperone-dependent amyloid assembly protects cells from prion toxicity.</a> Proc. Natl. Acad. Sci. USA. 105, 7206-7211 (2008).</li><li>Nagy, B., Costello, R., Csako, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Downward+blotting+of+proteins+in+a+model+based+on+apolipoprotein(a)+phenotyping.">Downward blotting of proteins in a model based on apolipoprotein(a) phenotyping.</a> Analytical Biochemistry. 231, 40-45 (1995).</li></ol>

SDD-AGE se informó por primera vez por Kryndushkin et al. 1, para el estudio de SDS-resistente a los complejos de la [PSI +] priones de levadura, y desde entonces ha encontrado un uso generalizado, tanto el estudio de priones y no agregados de priones 2-9. Sin embargo, la transferencia de las proteínas a una membrana de electroforesis siguiente en un gel de agarosa es problemático, y puede resultar en una imagen distorsionada mancha 5. Además, la técnica sumergida electroblotting más utilizado presenta limitaciones prácticas para el tamaño del gel y por lo tanto el número de muestras que pueden ser procesados. Hemos abordado estos problemas mediante el empleo de la transferencia a la baja capilar 10, un procedimiento simple que utiliza una pila de papeles secantes secos para la transferencia de las proteínas del gel a una membrana de nitrocelulosa. Transferencia capilar evita la distorsión y permite que los geles grandes para ser procesado fácilmente. Hay algunas cosas a considerar antes de usar SDD-AGE. Para las muestras de crudo (por ejemplo, lisados), inmunodetección de proteínas específicas es necesario. SDD-AGE no es totalmente desnaturalizar los complejos de proteínas de interés, por lo que la proteína (s) para ser detectados deben llevar una etiqueta de epítopo fuera de la región amiloidogénica. Lisados ​​general, se puede preparar como se haría para un normal SDS-PAGE, con dos diferencias importantes. En primer lugar, un mayor cuidado se debe tomar para evitar su degradación por proteolisis. Las condiciones parcialmente desnaturalizantes utilizados aquí no son suficientes para inactivar las proteasas, y también puede hacer las proteínas diana más susceptibles a la proteólisis. Use un cóctel de inhibidores de proteasa completa en al menos dos veces la concentración recomendada. En segundo lugar, el calentamiento de la muestra debe ser evitado. Si un control de monómero de todos los negativos que se desea, por ejemplo, para confirmar que el alto peso molecular de especies no se deben a modificaciones covalentes, de una incubación de 10 minutos a 95 ° C se puede utilizar, lo que restaurará la mayoría de los amiloides de la proteína monomérica.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>zymolyase 100T</td>
<td>&nbsp;</td>
<td>Seikagaku America</td>
<td>120493-1</td>
<td>&nbsp;</td>
<tr>
<td>Halt Protease Inhibitor Cocktail</td>
<td>&nbsp;</td>
<td>Thermo Scientific </td>
<td>78429</td>
<td>&nbsp;</td>
<tr>
<td>Hybond-C extra nitrocellulose</td>
<td>&nbsp;</td>
<td>Amersham</td>
<td>RPN303E</td>
<td>&nbsp;</td>
<tr>
<td>GB004 blotting paper</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>10427926</td>
<td>&nbsp;</td>
<tr>
<td>GB002 (3MM Chr) blotting paper</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>3030-917</td>
<td>&nbsp;</td>
<tr>
<td>Agarose (UltraPure)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15510-027</td>
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screening for amyloid aggregation by semi-denaturing detergent-agarose gel electrophoresis

Agregación amiloide se asocia con numerosas patologías mal plegamiento de proteínas y es la base de las propiedades de los priones infecciosos, que se auto-conformación de plantillas de las proteínas que se cree que tienen un papel beneficioso en los organismos inferiores. Amiloides han sido muy difíciles de estudiar debido a su insolubilidad y la heterogeneidad estructural. Sin embargo, la resolución de los polímeros de amiloide en función del tamaño y la insolubilidad de detergente ha sido posible gracias a Semi-desnaturalización de detergente-geles de agarosa (SDD-AGE). Esta técnica es encontrar amplio uso para la detección y caracterización de variantes conformacionales de amiloide. Este sentido, demuestran una adaptación de esta técnica que facilita su uso en aplicaciones a gran escala, como las pantallas de los priones y otras proteínas novela amiloidogénica. El nuevo SDD-AGE método utiliza la transferencia capilar para una mayor fiabilidad y facilidad de uso, y permite a cualquier tamaño de gel para ser acomodados. Por lo tanto, un gran número de muestras, preparadas a partir de células o proteínas purificadas, se pueden procesar simultáneamente la presencia de SDS-insoluble conformadores de proteínas etiquetadas.

Watch this Scientific Journal Video about Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis at JoVE.com

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

SDD-AGE es una técnica útil para la detección y caracterización de polímeros como el amiloide en las células. Aquí se demuestra una adaptación que hace que esta técnica susceptible de aplicaciones a gran escala.

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 ...

screening-for-amyloid-aggregation-semi-denaturing-detergent-agarose

Research

JoVE Journal

Biology

26.7K vistas.  Whitehead Institute for Biomedical Research. SDD-AGE es una técnica útil para la detección y caracterización de polímeros como el amiloide en las células. Aquí se demuestra una adaptación que hace que esta técnica susceptible de aplicaciones a gran escala.

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

Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis DGGE

Gradiente desnaturalizante electroforesis en gel (DGGE)

Investigación

Biología

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

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 separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method1. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 &deg;C during the gel run allows for the separation of unstructured DNA or RNA molecules.
In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.
In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol 1,2.

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.

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 ...

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 ...

Pouring and Running a Protein Gel by reusing Commercial Cassettes

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry and molecular biology researchers1-4. For laboratories that perform daily analyses of proteins, the cost of commercially available polyacrylamide gels (&tilde;$10/gel) can be considerable over time. To mitigate this cost, some researchers prepare their own polyacrylamide gels. Traditional methods of pouring these gels typically utilize specialized equipment and glass gel plates that can be expensive and preclude pouring many gels and storing them for future use. Furthermore, handling of glass plates during cleaning or gel pouring can result in accidental breakage creating a safety hazard, which may preclude their use in undergraduate laboratory classes. Our protocol demonstrates how to pour multiple protein gels simultaneously by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment. In addition, plastic gel cassettes are extremely resistant to breakage, which makes them ideal for undergraduate laboratory classrooms.

Our protocol demonstrates how to pour multiple protein gels at a time by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment.

Verter y ejecución de un gel de proteínas mediante la reutilización de casetes comerciales

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry ...

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 ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Selección de alto rendimiento para moduladores químicos de genes post-transcripcionalmente regulados

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

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 ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

Métodos de estudio cambios de agregación inherente de proteína con la edad en Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

Agregación del Receptor de superficie celular microscopia confocal revela a través espectroscopia de la correlación de imagen

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...

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 ...