Reconocemos Melissa Grunlan útil para los debates sobre la formación de SAM. Esta investigación fue financiada en parte por la Fundación Robert A. Welch (A-1585).

Preparación de los reactivos Preparar tampón PIPES (80 TUBOS mM, 1 mM MgCl2, 1 mM EGTA, ajustada a pH 6,8 con KOH). Alícuota y almacenar la glucosa oxidasa, catalasa y glucosa a -70 ° C y taxol a -20 ° C de acuerdo con los protocolos existentes. 1,2 alícuotas y la tubulina almacenar a -70 ° C de acuerdo con las instrucciones que se incluyen por el proveedor. Au microelectrodos patrón utilizando la litografía <ol><li> Corte de obleas de silicio en sustratos 1 cm &#39;1,5 cm con un escribano o un cortador de la oblea. Limpia los sustratos, colocándolos en un recipiente con acetona suficiente para cubrirlas y ultrasonidos durante 5 minutos. Sin permitir que el sustrato se seque, lávelos en acetona fresca, y luego enjuagar inmediatamente en nitrógeno con isopropanol y seco. </li><li> Fabricar microelectrodos Au sobre los sustratos limpios usando estándar, de tono positivo fotolitografía o litografía por haz de electrones, dependiendo del tamaño de las características deseadas. 3 En el diseño de la geometría del modelo, mantenga el diámetro de cada patrón de electrodos por debajo de 100 micrones para asegurar la cobertura de la PEG SAM (que se describe en la siguiente sección). Incluyen almohadillas de contacto (~ 300 mm 2) posición 300 a 500 mm del modelo con una línea de 1 mm de conectar el teclado con el electrodo (Fig. 1). Coloque los patrones litográficos cerca de la mitad del sustrato para permitir que el espacio alrededor del patrón para el montaje de la cámara de flujo. <a href="https://www.jove.com/files/ftp_upload/1390/1390fig1.jpg"><img border="0" src="/files/ftp_upload/1390/1390fig1thumb.jpg" alt="la figura 1" /></a> Figura 1. Geometría de la muestra. </li><li> Después del desarrollo de la estructura de la capa de resistir, el principio de una línea en la resistencia de la plataforma de contacto con el borde del sustrato con una pinza (Fig. 1). Tras la deposición de metales, el cero se permite el contacto eléctrico entre el electrodo y el borde del sustrato. Una cámara de deposición, de preferencia con dos fuentes, se utiliza para depositar la capa de metal. Los electrodos están formados por la deposición de 2 nm de Cr (de adhesión), seguido de 6 nm de Au, sin romper el vacío. Una microbalanza de cristal de cuarzo equipado dentro de la cámara se puede utilizar para medir el espesor de las películas durante la deposición. </li></ol> Preparar el SAM <ol><li> Precaliente el horno y en un área bien ventilada a 75 ° C. Prepare la solución silanización añadiendo 20 ml de tolueno a un vaso de vidrio de 250 ml. A continuación, añadir 1 ml de PEG-silano (es decir, 5% v / v) para el tolueno con una pipeta de plástico. Mezcle en el PEG-silano y pipeteando arriba y abajo 5 a 10 veces y luego se agita con la pipeta durante 30 segundos. </li><li> Colocar las muestras patrón en el vaso con el lado estampado mirando hacia arriba, asegurándose de que estén completamente sumergidos en la solución de tolueno PEG-silano y se espacian uniformemente en la parte inferior del vaso. Coloque el recipiente en el horno y hornear 18 a 21 horas a 75 ° C. Mientras que el SAM se está formando, preparar el contraelectrodo (ver abajo) y ensamblar los componentes adicionales necesarios para el resto de pasos. </li><li> Después de 18-21 horas en el horno, el tolueno se evapora dejando las muestras patrón de un líquido viscoso PEG-silano residuos. Agregar 20 ml de tolueno en el vaso y tomar las muestras durante 1-2 minutos. Quitar las muestras del vaso y enjuagar con tolueno, a continuación, isopropanol, y finalmente en seco con nitrógeno. En este punto, las muestras deberán mirada limpia y la capa de SAM debe ser invisible a simple vista. SAM preparado de esta forma se pueden utilizar hasta dos días cuando se almacena bajo condiciones de frío seco (25 ° C, humedad moderada). Infructuosos resultados de la formación de SAM en una capa de residuos nublado o desiguales tonos en la superficie. </li></ol> Fabricar el contraelectrodo En la configuración de la cámara de deposición mismo que se utiliza para la deposición de microelectrodos, hacer un contraelectrodo depositando 1 nm de Cr, seguido de 4 nm de Au en un 22 x 50 mm N º 1 y N º 0 cubreobjetos. El contraelectrodo debe ser casi transparente con un tinte de color gris claro o rosa. Guarde el contraelectrodo recubierta de arriba abajo en una placa de Petri para mantenerlo limpio. Tubulina polimeriza en MT <ol><li> Descongele etiqueta tubulina y tubulina rodamina y de inmediato se combinan en una proporción de 1:2 etiquetados con etiqueta para obtener brillantes MTs. Mezclar pipeteando arriba y abajo varias veces. Polimeriza la tubulina durante 20-40 minutos en un baño de agua caliente a 37 ° C. La longitud de la MT serán mayores para los tiempos de polimerización más. Mientras que la tubulina se polimeriza, preparar un tampón PIPES-taxol mediante la adición de paclitaxel a 20 mm en un tubo de microcentrífuga que contiene tampón PIPES. Mezclar el taxol y pipeteando arriba y abajo varias veces y agitación, y luego colocar el tubo en el baño se calienta a 37 ° C. Después de la polimerización, se diluye la MTs por 1 / 100 en caliente TUBOS taxol-buffer y el escudo de la luz. MTs preparado de esta manera se alarga más deel tiempo y el paquete, que puede ser reducida por dilución. </li><li> Preparar una mezcla de oxígeno 10 veces más basura (OS) mediante la combinación de 30 ml de tampón TUBOS frío (0-4 ° C), 5 ml de 50% 2-mercaptoetanol, 5 glucosa oxidasa ml, 5 ml de catalasa, glucosa y 5 ml. 2 de la glucosa debe añadirse pasado. Mezclar con la pipeta hacia arriba y abajo 3-5 veces después de agregar cada componente. Coloque la mezcla de OS en el hielo, sino que se mantendrá vigente por aproximadamente 2 horas. </li><li> Verifique que la polimerización MT fue un éxito y que la MT son estables en varios minutos de excitación de fluorescencia por imágenes de los preparados MTs con 1 mezcla de OS. Para obtener imágenes de los MTs preparado de esta manera, asegúrese de usar una combinación de excitación / emisión de filtro adecuado para la fluorescencia de rodamina. Determinar la dilución de trabajo óptima para los filamentos de imágenes individuales por diluyendo aún más el sistema multilateral de 1 / 10 a 1 / 100 con tampón PIPES y las imágenes con 1 mezcla de OS. </li><li> Preparar una cámara de flujo colocando un grande (22 x 50 mm) y pequeño (22 x 22 mm) cubreobjetos con dos pedazos de cinta de doble cara ponen 2-3 mm. La cinta es fácil de cortar en tiras, colocándolo en un portaobjetos de vidrio y el corte con una cuchilla de afeitar. Introducir unos pocos microlitros de MTs en la celda de flujo utilizando una micropipeta. Para imágenes de gran aumento, mucho trabajo los objetivos de la distancia puede ser necesario para obtener imágenes de la superficie superior de la cámara de flujo. La distancia desde la superficie del cubreobjetos / muestra a la superficie superior de la cámara de flujo se determina por el grosor de la cinta y debe ser 60 a 100 mm. </li></ol> Patrón de MTs mediante electroforesis <ol><li> Preparar una cámara de electroforesis mediante la colocación de dos finas tiras de cinta de doble cara en la muestra patrón, SAM recubiertos de tal manera que el electrodo se encuentra entre las tiras de conexión con la ruta perpendicular a la cinta (Fig. 2). Coloque el contraelectrodo en la cinta de doble cara con la parte de metal que tocan la cinta de tal manera que el borde de los aleros de la muestra el contraelectrodo en alrededor de 3 mm (Fig. 2). Corte varias longitudes de 15 cm de alambre de cobre con aislamiento magnético y tira de 1 cm del aislamiento de los dos extremos. Conecte un cable a la zona expuesta Au del sustrato con una pequeña gota de pintura de plata, mientras que teniendo cuidado de no hacer contacto eléctrico con el contraelectrodo (esto se puede comprobar con un multímetro). Conecte un segundo cable al contraelectrodo con pintura plateada. Cables conectados con la pintura de plata puede ser más seguro para las muestras con cinta adhesiva. <a href="https://www.jove.com/files/ftp_upload/1390/1390fig2.jpg"><img src="/files/ftp_upload/1390/1390fig2thumb.jpg" border="0" alt="la figura 2" /></a> Figura 2. Flujo conjunto de células de un microscopio de fluorescencia invertido. </li><li> Introducir MTs a la dilución deseada utilizando tampón PIPES combinado con el sistema operativo a 1x concentración final. Imagen de 20X y localizar el patrón en la oblea de silicio antes de aplicar un voltaje. Opcionalmente, la cámara de flujo pueden ser sellados con esmalte de uñas para evitar que el flujo de fluidos y la migración de MT en el plano de la cámara de flujo. </li><li> Aplique una tensión de hasta 1 V y observar la migración de las MTs. Voltajes más bajos (hasta cerca de 0.5 V) se puede utilizar si atrapando reversible de MTs se desea, sin embargo esto requiere de sellado de la cámara de flujo para evitar la deriva lateral. Esmalte de uñas que funciona bien para el sellado de los extremos abiertos de la cámara de flujo. Con tensiones superiores a unos 0,7 V, algunos de adhesión MT puede ser visto en el patrón, aunque será débil. Tensiones superiores a alrededor de 0,9 V, la migración de MT será más rápido, y la adhesión será más fuerte. Por encima de alrededor de 1,2 V de la probabilidad de inducción de la electrólisis (formación de burbujas de gas) y también para la destrucción de los microelectrodos se incrementa significativamente. La migración puede ser rastreado mediante el ajuste del plano vertical del foco del microscopio. Si se lleva a cabo correctamente, el MTS se localizan en la superficie de los electrodos. </li></ol>

<ol>
	<li>Rai-Choudhury, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Microlithography, Micromachining, and Microfabrication: Microlithography. , (1997).</li></ol>

The authors have nothing to disclose.

La migración de MT es fácil de visualizar mediante microscopía de fluorescencia debido a su brillo y la fluorescencia de fondo bajo. El método es aplicable en general a los patrones de biomoléculas, ya que sólo se requiere que las biomoléculas ser inducidos a realizar un cargo neto por el ajuste adecuado del pH. Electroósmosis se evita en esta configuración, porque no hay superficies cargadas, tales como las paredes de sílice utilizado en electroforesis capilar. Varias modificacion...

No. 1 de vidrio de borosilicato cubreobjetos (22 x 50 mm) fueron adquiridos de VWR. Pulido grado electrónico de tipo p &lt;111&gt; obleas de silicio fueron adquiridos de Addison Engineering, Inc. de alta pureza pintura de plata fue obtenida de Structure Probe, Inc. (Catálogo # 5001 AB). Aislado de cobre fino calibre imán (~ 0,08 mm (0,003 pulgadas) de diámetro) se puede obtener de las compañías de componentes electrónicos.

antifouling self-assembled monolayers on microelectrodes for patterning biomolecules

Se presenta un procedimiento para la formación de un poli (etilenglicol) (PEG) trimetoxisilano auto-ensambladas monocapa (SAM) en un substrato de silicio con microelectrodos de oro. El PEG-SAM se forma en una etapa de montaje simple y evita incrustaciones en las superficies de silicio y oro. El SAM se utiliza para microelectrodos abrigo estampado con el estándar, de tono positivo litografía. El uso de los microtúbulos como ejemplo, se aplica un voltaje de DC para inducir la migración electroforética al electrodo SAM recubiertos de una manera reversible. Una cámara de flujo se utiliza para obtener imágenes de la migración y los patrones de electroforesis de microtúbulos<em&gt; In situ</em&gt; Mediante microscopía de epifluorescencia. Este método es generalmente aplicable a los patrones de biomoléculas, ya que emplea la electroforesis para inmovilizar las moléculas objetivo y por lo tanto no requiere de interacciones moleculares específicas. Además, evita los problemas encontrados al intentar patrón de las moléculas de SAM directamente utilizando técnicas litográficas. La compatibilidad con litografía por haz de electrones permite a este método que se utilizará para las biomoléculas patrón en la nanoescala.

Watch this Scientific Journal Video about Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules at JoVE.com

Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules

Se presenta un procedimiento para la formación de un poli (etilenglicol) auto-ensambladas monocapa (PEG-SAM) en un substrato de silicio con microelectrodos de oro. El PEG-SAM se forma en un solo paso y evita la contaminación biológica en las superficies de silicio y oro. Electroforesis se utiliza para modelar biomoléculas a la nanoescala.

Antifouling monocapas auto-ensambladas de microelectrodos de Biomoléculas Patrones

We present a procedure for forming a poly(ethylene glycol) (PEG) trimethoxysilane self-assembled monolayer (SAM) on a silicon substrate with gold ...

antifouling-self-assembled-monolayers-on-microelectrodes-for

Research

JoVE Journal

Biology

11.4K vistas.  Texas A&M University (TAMU). Se presenta un procedimiento para la formación de un poli (etilenglicol) auto-ensambladas monocapa (PEG-SAM) en un substrato de silicio con microelectrodos de oro. El PEG-SAM se forma en un solo paso y evita la contaminación biológica en las superficies de silicio y oro. Electroforesis se utiliza para modelar biomoléculas a la nanoescala.

Video: Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules

Patterning Cells on Optically Transparent Indium Tin Oxide Electrodes

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular constructs serving as in vitro mimics of native tissues.  In this study, photolithography and wet etching techniques were used to fabricate individually addressable indium tin oxide (ITO) electrodes on glass substrates.  The glass substrates containing ITO microelectrodes were modified with poly(ethylene glycol) (PEG) silane to make them protein and cell resistive.  Presence of insulating PEG molecules on the electrode surface was verified by cyclic voltammetry employing potassium ferricyanide as a redox reporter molecule.  Importantly, the application of reductive potential caused desorption of the PEG layer, resulting in regeneration of the conductive electrode surface and appearance of typical ferricyanide redox peaks.    Application of reductive potential also corresponded to switching of ITO electrode properties from cell non-adhesive to cell-adhesive. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.  Micropatterning of several cell types was demonstrated on these substrates.   In the future, the control of the biointerfacial properties afforded by this method will allow to engineer cellular microenvironments through the assembly of three or more cell types into a precise geometric configuration on an optically transparent substrate.

Non-fouling PEG silane monolayer was desorbed from individually addressable ITO electrodes on glass by application of a reductive potential. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.

Las células de patrón indio electrodos ópticamente transparente de óxido de estaño

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular ...

Investigación

Biología

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

Los patrones de las células madre embrionarias usando el Flip Chip Bio

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

Sistema Automatizado de Soltero mediciones de fluorescencia de la molécula de superficie inmovilizada Biomoléculas

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

Perfiles de transcripción de los organismos unicelulares de los cardiomiocitos de ratón adulto

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

We developed computational de novo protein design methods capable of tackling several important areas of protein design. To disseminate these methods we present Protein WISDOM, an online tool for protein design (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>). Starting from a structural template, design of monomeric proteins for increased stability and complexes for increased binding affinity can be performed.

SABIDURÍA Proteína: Un banco de trabajo para<em&gt; En silico</em&gt;<em&gt; De novo</em&gt; Diseño de biomoléculas

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific ...

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. 
We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisi&#230;, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.

Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

Internalización y Observación de fluorescente Biomoléculas en microorganismos que viven a través de electroporación

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Medición y Análisis de la producción de ácido extracelular para Determinar glicolítica Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

El aislamiento de regiones genómicas específicas e identificación de moléculas asociadas por enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

Cuantificación de proteína específica lisina acetilación y Succinylation estequiometría usando Spectrometry total de la adquisición de datos-independiente

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

SA-&amp;#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

Ensayo de cribado basado en sA-Galactosidasa para la identificación de medicamentos senoterapéuticos

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...