Los autores desean agradecer a: Dr. Frank Jiang, Dr. David Lin, el Dr. Bernard Yurke y el Dr. Uday Chippada por sus contribuciones en el desarrollo de la tecnología de gel de ADN; Norell Hadzimichalis, Smit Shah, Kimberly Peterman, Robert Arter por sus comentarios y ediciones de este manuscrito; fuentes de financiación, incluida la Comisión de Nueva Jersey en la médula espinal de Investigación (Grant # 07A-019-SCR1, NAL) y Nueva Jersey Instituto de Neurociencias (MLP); y editores de Ingeniería de Tejidos, Parte A para la autorización de reimprimir las figuras 2 y 4 y Biomateriales permiso para reimprimir la figura 3.

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Los autores no tienen nada que revelar.

La capacidad de los geles de ADN para suavizar o endurecer antes y después de la adhesión celular las hace un modelo ideal para estudiar el papel de la rigidez del tejido dinámico en función de la célula. Todos los tres diseños se han utilizado en estudios mecánicos y biológicos. Sin embargo, los tres diseños tienen elasticidades similares en diversos porcentajes de reticulación, lo que indica la longitud de reticulación no influye elasticidad del gel de ADN (Tabla 2). En contraste, la concen...

Sustratos estáticos y dinámicos son dos categorías de biomateriales que se desarrollaron para estudiar los efectos de la elasticidad de los tejidos o rigidez en la función celular. Sustratos estáticas son incapaces de cambiar sus propiedades físicas después de que se fabrican y / o una vez se siembran las células. Poliacrilamida (PA) geles fueron los primeros sustratos bidimensionales estáticas, que se sintetizaron las investigaciones mecanobiología 5,17. Geles de PA son fáciles de preparar, de bajo costo, versátil, y puede ser fabricado con una amplia gama de módulos elásticos. Aunque estas ventajas técnicas hacen PA geles un sustrato general aplicada, sustratos estáticas no son indicativos de la naturaleza dinámica de la matriz extracelular (MEC) y que rodea entorno celular in vivo. Por ejemplo, el ECM sufre alteraciones de rigidez como resultado de una lesión, el desarrollo, o la enfermedad. Por lo tanto sustratos dinámicos son favorecidos como los modelos de sustrato que imitan los tejidos en los estudios mecanobiología <sup&gt; 22,24,25. Numerosos sintética, bidimensional biomateriales, tridimensionales, estáticas y dinámicas naturales se han desarrollado para imitar la rigidez del tejido 1,3,6,16,23,26. Algunos sustratos dinámicos requieren calor, UV, la corriente eléctrica, los iones, y los cambios de pH para alterar sus propiedades mecánicas 2,4,7,8,12,15,16, pero estos estímulos pueden restringir bio-aplicación del hidrogel. Hidrogeles de poliacrilamida reticulada de ADN-ADN (geles) son sustratos elásticos bidimensionales dinámicos. Entrecruzamientos de ADN permiten la modulación temporal, espacial, y reversible de la rigidez en gel de ADN mediante la adición de ADN de cadena sencilla (ssDNA) a los medios o tampón 9-11,13,14,18,21. A diferencia de los geles dinámicas mencionadas anteriormente donde se aplican los estímulos para la modulación de elasticidad, los geles de ADN se basan en la difusión de ssDNA aplicada para la alteración de la elasticidad. Por lo tanto, la superficie del gel superior, donde se cultivan las células, es la primera área modulada porque el o tasamodulación f elasticidad es dependiente del espesor de gel. Geles de ADN son similares a sus homólogos de gel de PA en que tienen un esqueleto de poliacrilamida, sin embargo, las reticulaciones-bis acrilamida se reemplazan con reticulaciones compuestas de ADN (Figura 1). Dos ssDNAs (SA1 y SA2) se hibridan con una cadena reticulante (L2) para compensar los entrecruzamientos de ADN del gel. SA1 y SA2 tienen secuencias distintas que ambos contienen una modificación Acrydite en el extremo 5 &#39;para su incorporación efectiva a la red PA. Para la preparación de los geles, SA1 y SA2 se polimerizan individualmente en una cadena principal de PA y, posteriormente, el SA1 SA2 polimerizado y se mezclan juntos. L2, el agente de reticulación, se añade a la SA1 y SA2 mezcla. La secuencia de bases es complementaria a L2 ambas secuencias SA1 y SA2 y L2 se hibrida con SA1 SA2 más para formar los entrecruzamientos de ADN. , Elasticidad del gel de ADN inicial es determinado por ambas concentraciones L2 y reticulación (Tablas 1 </strong&gt; y 2). Geles de ADN que contienen cantidades estequiométricas iguales de L2, SA1, SA2 y son los geles más rígidas porque SA1 y SA2 son 100% reticulado por L2 (designado como 100% geles). Concentraciones más bajas de L2 resultado en un menor porcentaje de reticulación del ADN y, por tanto, geles más blandos de ADN. Geles tan bajas como 50% reticulado (designado como 50% en geles) se han construido 9-11. <img alt="Figura 1" src="/files/ftp_upload/51323/51323fig1highres.jpg" /> Figura 1. reticulación de gel de ADN y uncrosslinking esquemática 9-11,13,14,18,21 Paso 1:. SA1 (rojo) y SA2 (azul) se polimerizan de forma individual en una columna vertebral de poliacrilamida (negro). Después de la polimerización, soluciones polimerizadas SA1 y SA2 se mezclan juntos. Paso 2: L2 (verde) se añade y se hibrida con SA1 SA2 más para formar los entrecruzamientos del gel. Paso 3: R2 hibrida con tél punto de apoyo de la L2. Paso 4: hibridación de punto de apoyo de R2 impulsa la descompresión de la L2 de SA1 y SA2. A diferencia de los geles de PA, geles de ADN pueden endurecerse y suavizar después de la síntesis. Por esa razón, las células cultivadas en geles de ADN pueden ser sometidos a alteraciones de rigidez dinámica. Para endurecer geles de células adherente, L2 puede ser añadido al medio de cultivo de los geles porcentuales bajos para aumentar el porcentaje de reticulaciones. Para suavizar los geles de células adherente, L2 se puede quitar para disminuir el porcentaje de entrecruzamientos 10,13,21. L2 tiene una secuencia de punto de apoyo adicional en el extremo 3 &#39;para permitir L2 para uncrosslink de SA1 y SA2 (Tabla 1). La eliminación de L2 se lleva a cabo mediante hibridación de una hebra de reversión llamado R2. R2 es complementaria a la longitud completa de L2 y se hibrida primero con el punto de apoyo L2. Hibridación propulsa el punto de apoyo de descompresión de L2 de SA1 y SA2, que elimina la reticulación y reduce la rigidez de gel. En el presente informe yvideo, paso a paso las instrucciones se proporcionan para la preparación de rigidización y suavizar los geles de ADN. Si bien se describen preparaciones de gel 100% y 80%, este protocolo se puede adaptar para crear geles de ADN de otros porcentajes reticulados inicial y final. En general, 100% y 80% en geles se preparan, inmovilizan sobre cubreobjetos de vidrio, funcionalizados, y se sembraron con células. L2 se añade a los medios de 80% de geles y R2 se añade a los medios de comunicación de 100% geles, 48 ​​hr después de la siembra. La adición de L2 a los medios endurece 80% a 100% geles reticulados, mientras que la adición de R2 a los medios suaviza 100% en geles de 80% reticulado. Geles endurecidas se designan como 80 → 100% geles y geles ablandados se designan como 100 → 80% de geles en el texto. Para controlar o geles estáticas, ssDNA que consiste en Ts o Como se entrega a otro conjunto de 100% y 80% en geles. Después de un mínimo de dos días siguientes modulación de elasticidad, las células pueden ser procesados ​​y analizados. <table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page = &quot;always&quot;&gt; <tbody align="center"><tr><td rowspan="2"></td><td rowspan="2"> Entrecruzamiento de ADN </td><td rowspan="2"> # De bases </td><td> Secuencia (punto de apoyo) </td><td rowspan="2"> Modificación </td><td rowspan="2"> Temperatura de fusión (T m, ° C) </td><td rowspan="2"> Comentarios </td></tr><tr><td> 5 &#39;→ 3&#39; </td></tr><tr><td rowspan="4"> Diseño 1 </td><td> SA1 </td><td> 10 </td><td> GCA CCT TTG C </td><td> 5 &#39;Acrydite </td><td> 34.9 </td><td></td></tr><tr><td> SA2 </td><td> 10 </td><td> GTC AGA ATG A </td><td> 5 &#39;Acrydite </td><td> 23.6 </td><td></td></tr><tr><td> L2 </td><td> 30 </td><td> TCA TTC TGA C GC AAA GGT GC T CTA CAC TTG </td><td></td><td> 56 </td><td>Una secuencia de 10 pb punto de apoyo está incluido. </td></tr><tr><td> R2 </td><td> 30 </td><td> CAA GTG TAG CGC ACC TTT GCG TCA GAA TGA </td><td></td><td></td><td> R2 es complementaria a L2 </td></tr><tr><td rowspan="4"> Diseño 2 </td><td> SA1 </td><td> 14 </td><td> CGT GGC ATA GGA CT </td><td> 5 &#39;Acrydite </td><td> 46.9 </td><td></td></tr><tr><td> SA2 </td><td> 14 </td><td> GTT TCC CAA TCA GA </td><td> 5 &#39;Acrydite </td><td> 40.2 </td><td></td></tr><tr><td> L2 </td><td> 40 </td><td> TCT GAT TGG GAA AC A GTC CTA TGC CAC T GT TAC CTT CAT C </td><td></td><td> 65.9 </td><td> A 12 pb secuencia de punto de apoyo está incluido. </td></tr><tr><td> R2 </td><td> 40 </td><td> GAT GAA GGT AAC CGT GGC ATA GGA CTG TTT CCC AAT CAG A </td><td></td><td> 65.9 </td><td> R2 es complementaria a L2 </td></tr><tr><td rowspan="3"> Design 3 </td><td> SA1 </td><td> 20 </td><td> ACG GAG GTG TAT GCA ATG TC </td><td> 5 &#39;Acrydite </td><td> 55 </td><td></td></tr><tr><td> SA2 </td><td> 20 </td><td> CAT GCT TAG GGA CGA CTG GA </td><td> 5 &#39;Acrydite </td><td> 56.6 </td><td></td></tr><tr><td> L2 </td><td> 40 </td><td> TCC AGT CGT CCC TAA GCA TG T ACA TTG CAT ACA CCT CCG T </td><td></td><td> 68.8 </td><td> Punto de apoyo no está incluido. </td></tr><tr><td rowspan="2"> Control de </td><td rowspan="2"> Control de </td><td rowspan="2"> 20-40 </td><td> AAA AAA (etc.) o </td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2"></td></tr><tr><td> TTT TTT (etc.) </td></tr></tbody></table> Cuadro 1 secuencias de base para ssDNA 9-11,13,14,18,21. Celular y estudios mecánicos han utilizados varios ddiseños de reticulación ifferent para generar geles de ADN con una gama de propiedades mecánicas estáticas y dinámicas. Los parámetros modulados en el diseño de reticulación son secuencia de bases y longitud de la secuencia o la longitud de reticulación. Negrita y cursiva ilustran base de sincronización entre SA1 y L2 y entre SA2 y L2, respectivamente. <table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody align="center"><tr><td rowspan="2"></td><td colspan="8"> Diseño </td></tr><tr><td colspan="3"> 1 </td><td colspan="3"> 2 </td><td colspan="2"> 3 </td></tr><tr><td> Concentración de acrilamida (%) </td><td colspan="3"> 10 </td><td colspan="3"> 10 </td><td> 10 </td><td> 4 </td></tr><tr><td> SA1 SA2 más hibridado con L2 (% reticulado) </td><td> 50 </td><td> 80 </td><td> 100 </td><td> 50 </td><td> 80 </td><td> 100 </td><td> 100 </td><td> 100 </td></tr><tr><td> &lt;strong&gt; Elasticidad (kPa, media ± SEM) </td><td> 6,6 ± 0,6 </td><td> 17,1 ± 0,8 </td><td> 29,8 ± 2,5 </td><td> 5,85 ± 0,62 </td><td> 12.67 ± 1.33 </td><td> 22.88 ± 2.77 </td><td> 25,2 ± 0,5 </td><td> 10,4 ± 0,6 </td></tr></tbody></table> Tabla 2. módulo de Young (E) de geles de ADN. 9-11,13,14,18,21 concentración de acrilamida, el porcentaje de reticulación, y la longitud de reticulación puede ser modulada en geles de ADN. Diseños 1, 2 y 3 tienen 20, 28, y 40 pb longitudes de reticulación, respectivamente. 100% de geles para todos los diseños tienen módulos similares, indicando la longitud de reticulación no afecta a la elasticidad del gel. Sin embargo, las variaciones en la concentración de acrilamida alteran elasticidad del gel del ADN.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>ssDNA</td>
			<td>Integrated DNA Technologies (Coralville, Iowa) idtdna.com</td>
			<td></td>
			<td>Do not vortex ssDNA. Gentle invert the vial and/or pipette solution to mix.</td>
		</tr>
		<tr>
			<td>PBS with calcium and magnesium</td>
			<td></td>
			<td></td>
			<td>Any brand.</td>
		</tr>
		<tr>
			<td>100X Tris-EDTA buffer (TE buffer)</td>
			<td>Sigma-Aldrich (St. Loius, MO) sigmaldrich.com</td>
			<td>T9285</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Tris-Borate-EDTA buffer (TBE buffer)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>93290</td>
			<td>TBE is a reproductive toxin.</td>
		</tr>
		<tr>
			<td>40% Acrylamide solution</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>BP14021</td>
			<td>Acrylamide is a toxin.</td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>A3678</td>
			<td>Prepare a 2% solution in TE buffer. APS is a toxin and irratant.</td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>T9281</td>
			<td>Prepare a 20% solution in TE buffer. TEMED is flammable, a corrosive, and a toxin.</td>
		</tr>
		<tr>
			<td rowspan="2">12-mm diameter round coverglass</td>
			<td rowspan="2">Fisher Scientific (Pittsburg, PA) fishersci.com</td>
			<td rowspan="2">12-545-82</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>Norland optical adhesive 72</td>
			<td>Norland Products (Cranbury, NJ) norlandprod.com</td>
			<td>NOA72</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">24-well tissue culture plate</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">Any brand.</td>
		</tr>
		<tr>
			<td rowspan="2">Microcentrifuge tubes</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">Any brand.</td>
		</tr>
		<tr>
			<td>Sulfo-SANPAH</td>
			<td>ProteoChem or Thermo Fisher, (Rockland, IL) proteochem.com or thermofisher.com</td>
			<td>C111 or 22589</td>
			<td>Prepare a 0.315 mg/ml solution in water immediately before use. Dissolve at 37&#176;C and filter sterilze. It is normal to observe undisolved sulfo-SANPAH in the filter. Sulfo-SANPAH is light sensitive and, therefore, the solution should be protect from light until UV exposure.</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine (PDL)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>P6407</td>
			<td>Prepare a 0.2 mg/ml solution in water and filter sterilize.</td>
		</tr>
		<tr>
			<td>Collagen Type I</td>
			<td>Affymetrix (Santa Clara, CA) affymetrix.com</td>
			<td>13813</td>
			<td>Prepare a 0.2 mg/ml solution in 0.2 N acetic acid. Solution needs to remain cold at all times to avoid polymerization. Acetic acid is a flammable, toxic, and corrosive.</td>
		</tr>
		<tr>
			<td>22 X 60 cover glass</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>12-544-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Positive-displacement pipette</td>
			<td>Gilson, Inc (Middletown, WI) gilson.com</td>
			<td>F148504</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat block</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>11-718</td>
			<td></td>
		</tr>
		<tr>
			<td>UV light source</td>
			<td></td>
			<td></td>
			<td>Place gels as close as possible to the UV light. UV light can cause skin or eye injury.</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td></td>
			<td></td>
			<td>Any brand.</td>
		</tr>
		<tr>
			<td>Nitrogen gas</td>
			<td>GTS-Welco (Flemington, NJ) www.praxairmidatlantic.com/</td>
			<td>NI 5.0UH-R</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of dna-crosslinked polyacrylamide hydrogels

Mecanobiología es un área científica emergente que aborda el papel fundamental de las señales físicas en la dirección de la morfología y la función celular. Por ejemplo, el efecto de la elasticidad de los tejidos en función de la célula es un área importante de la investigación mecanobiología porque la rigidez del tejido modula con la enfermedad, el desarrollo, y las lesiones. Materiales estáticos que imitan tejidos o materiales que no pueden alterar la rigidez una vez que se siembran las células, se utilizan principalmente para investigar los efectos de la rigidez del tejido en las funciones celulares. Si bien la información obtenida de los estudios estáticos es valiosa, estos estudios no son indicativos de la naturaleza dinámica del microambiente celular in vivo. Para abordar mejor los efectos de la rigidez dinámica en función de la célula, se desarrolló un sistema de hidrogel de poliacrilamida ADN reticulado (geles de ADN). A diferencia de otros sustratos dinámicos, geles de ADN tienen la capacidad de aumentar o disminuir la rigidez después de la fabricación y sin estímulos. Geles de ADN consisten en ADN crossltintas que se polimerizan en un esqueleto de poliacrilamida. Adición y eliminación de enlaces cruzados a través de la entrega de ADN de cadena sencilla permite temporal, espacial y de control reversible de la elasticidad del gel. Hemos demostrado en los informes anteriores que la modulación dinámica de la elasticidad del gel del ADN influye en el comportamiento de fibroblastos y la neurona. En el presente informe y el vídeo, ofrecemos un esquema que describe los mecanismos de reticulación de gel de ADN y las instrucciones paso a paso sobre los geles de ADN preparación.

Antes de nuestros estudios, se observaron las interacciones célula-ECM en biomateriales compatibles estáticas o en sustratos dinámicos irreversibles y unidireccionales. Estos sustratos no reflejan con exactitud la naturaleza dinámica del microambiente celular. Nuestro trabajo cambia los paradigmas técnicos existentes al proporcionar un modelo más fisiológico para el estudio de las interacciones célula-ECM de ablandamiento y endurecimiento biomateriales dinámicos. En estudios anteriores, se analizaron los efecto...

Watch this Scientific Journal Video about Preparation of DNA-crosslinked Polyacrylamide Hydrogels at JoVE.com

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Nuestro laboratorio ha desarrollado hidrogeles de poliacrilamida de ADN-reticulada, un sistema dinámico de hidrogel, para entender mejor los efectos de la modulación de la rigidez del tejido en función de la célula. Aquí, ofrecemos esquemas, descripciones y protocolos para preparar estos hidrogeles.

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, ...

preparation-of-dna-crosslinked-polyacrylamide-hydrogels

Research

JoVE Journal

Biology

14.5K vistas.  JFK Medical Center.  Nuestro laboratorio ha desarrollado hidrogeles de poliacrilamida de ADN-reticulada, un sistema dinámico de hidrogel, para entender mejor los efectos de la modulación de la rigidez del tejido en función de la célula. Aquí, ofrecemos esquemas, descripciones y protocolos para preparar estos hidrogeles. 

Video: Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Horizontal Slice Preparation of the Retina

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of retinal neurons, such as amacrine cells, expand their dendrites horizontally, so that the morphology of the cells is supposed to be severely damaged in the vertical slices. In the whole-mount preparation, especially for patch-clamp recordings, retinal neurons in the middle layer are not easily accessible due to the extensive coverage of glial cell (Mueller cell) s endfeets. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact. The slice was made horizontally at the inner layer of the retina using a vibratome slicer after the retina was embedded in the low-temperature melting agarose gel. In this horizontal slice preparation of the retina, we studied the function of retinal neurons compared with their morphology, by using patch-clamp recording, calcium imaging technique, immunocytochemistry, and single-cell RT-PCR.

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact.

Preparación de corte horizontal de la retina

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of ...

Investigación

Biología

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

Preparación de los cultivos disociados ratón neuronas corticales

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

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 ...

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 ...

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

La encapsulación celular en hidrogeles 3D para ingeniería de tejidos

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

El estudio de los efectos de la matriz de rigidez en la función celular usando como base la acrilamida-hidrogeles

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer 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 ...

Preparation of Quality Inositol Pyrophosphates

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells are inositol pentakisphosphate (IP5) and inositol hexakisphosphate (phytic acid or IP6). IP5 and IP6 are the precursors of inositol pyrophosphate molecules that contain one or more pyrophosphate bonds1. Phosphorylation of IP6 generates diphoshoinositolpentakisphosphate (IP7 or PP-IP5) and bisdiphoshoinositoltetrakisphosphate (IP8 or (PP)2-IP4). Inositol pyrophosphates have been isolated from all eukaryotic organisms so far studied. In addition, the two distinct classes of enzymes responsible for inositol pyrophosphate synthesis are highly conserved throughout evolution2-4.
The IP6 kinases (IP6Ks) posses an enormous catalytic flexibility, converting IP5 and IP6 to PP-IP4 and IP7 respectively and subsequently, by using these products as substrates, promote the generation of more complex molecules5,6. Recently, a second class of pyrophosphate generating enzymes was identified in the form of the yeast protein VIP1 (also referred as PP-IP5K), which is able to convert IP6 to IP7 and IP87,8.
Inositol pyrophosphates regulate many disparate cellular processes such as insulin secretion9, telomere length10,11, chemotaxis12, vesicular trafficking13, phosphate homeostasis14 and HIV-1 gag release15. Two mechanisms of actions have been proposed for this class of molecules. They can affect cellular function by allosterically interacting with specific proteins like AKT16. Alternatively, the pyrophosphate group can donate a phosphate to pre-phosphorylated proteins17. The enormous potential of this research field is hampered by the absence of a commercial source of inositol pyrophosphates, which is preventing many scientists from studying these molecules and this new post-translational modification. The methods currently available to isolate inositol pyrophosphates require sophisticated chromatographic apparatus18,19. These procedures use acidic conditions that might lead to inositol pyrophosphate degradation20 and thus to poor recovery. Furthermore, the cumbersome post-column desalting procedures restrict their use to specialized laboratories.
In this study we describe an undemanding method for the generation, isolation and purification of the products of the IP6-kinase and PP-IP5-kinases reactions. This method was possible by the ability of polyacrylamide gel electrophoresis (PAGE) to resolve highly phosphorylated inositol polyphosphates20. Following IP6K1 and PP-IP5K enzymatic reactions using IP6 as the substrate, PAGE was used to separate the generated inositol pyrophosphates that were subsequently eluted in water.

Inositol pyrophosphates play an important role in human pathologies such cancer, diabetes and obesity; however, the exact mechanism of action is a matter of dispute. The lack of commercially available inositol pyrophosphates renders detailed studies problematic. Here we describe a simple protocol to produce and isolate milligrams of inositol pyrophosphates.

Preparación de la Calidad pirofosfatos Inositol

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells ...

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

La estimulación mecánica de condrocitos-agarosa hidrogeles

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET Imaging en hidrogeles tridimensionales

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...