Reconhecemos Melissa Grunlan para discussões úteis sobre a formação de SAM. Esta pesquisa foi suportada em parte pela A. Robert Welch Foundation (A-1585).

Preparação de reagentes Prepare TUBOS tampão (80 mM PIPES, 1mM MgCl 2, 1 mM EGTA, ajustado para pH 6,8 com KOH). Alíquotas e armazenar glicose oxidase, catalase e glicose a -70 ° C e taxol a -20 ° C de acordo com protocolos existentes. 1,2 alíquotas e tubulina armazenar a -70 ° C de acordo com as instruções fornecidas pelo fornecedor. Microeletrodos padrão de Au utilizando litografia <ol><li> Corte em pastilhas de silício 1,5 1 cm &#39;substratos cm, utilizando um escriba ou cortador de bolacha. Limpe os substratos, colocando-os num copo com acetona o suficiente para cobri-los e sonicate por 5 minutos. Sem permitir que os substratos para secar, lave-os em acetona fresco, logo em seguida enxágüe em isopropanol e seco usando nitrogênio. </li><li> Au fabricar microeletrodos nos substratos limpos usando o padrão, fotolitografia tom positivo ou litografia por feixe de elétrons, dependendo do tamanho das características desejadas. 3 Ao projetar a geometria padrão, manter o diâmetro de cada padrão de eletrodo inferior a 100 microns para garantir a cobertura pela PEG SAM (descrito na próxima seção). Incluem suportes de contato (~ 300 mm 2) posicionados 300-500 mm do padrão com uma linha de 1 milímetro de conectar o teclado com o eletrodo (Fig. 1). Coloque os padrões litográficos perto do meio do substrato para permitir o espaço em torno do padrão para a montagem da câmara de fluxo. <img border="0" src="/files/ftp_upload/1390/1390fig1thumb.jpg" alt="figura 1" /> Figura 1. Geometria da amostra. Por favor, <a href="https://www.jove.com/files/ftp_upload/1390/1390fig1.jpg">clique aqui</a> para ver uma versão ampliada da figura 1. </li><li> Após o desenvolvimento do padrão na camada de resistir, um arranhão na linha de resistir a partir do bloco de contato para a borda do substrato usando uma pinça (Fig. 1). Após a deposição de metal, do zero vai permitir o contato elétrico entre o eletrodo ea borda do substrato. A câmara de deposição, de preferência com duas fontes, é utilizado para depositar a camada metálica. Os eletrodos são formados pela deposição de 2 nm de Cr (por adesão), seguido de 6 nm de Au, sem quebra de vácuo. A microbalança de cristal de quartzo equipado dentro da câmara pode ser usada para medir a espessura dos filmes durante a deposição. </li></ol> Preparar o SAM <ol><li> Pré-aqueça o forno e em uma área bem ventilada a 75 ° C. Preparar a solução de silanização, adicionando 20 mL de tolueno para um copo de vidro de 250 ml. Em seguida, adicione 1 mL PEG-silano (ou seja, 5% v / v) para o tolueno, utilizando uma pipeta de plástico. Misture o PEG-silano bem pipetando cima e para baixo 5 a 10 vezes e depois mexendo com a pipeta de 30 segundos. </li><li> Coloque as amostras padrão no recipiente com o lado virado para cima padrão, certificando-se que eles são completamente submersas na solução de PEG-silano tolueno e são espaçadas ao longo do fundo do copo. Colocar o copo no forno e asse 18-21 horas a 75 ° C. Enquanto o SAM está se formando, preparar o counterelectrode (veja abaixo) e montar os componentes adicionais necessários para as etapas restantes. </li><li> Após 18-21 horas no forno, o tolueno deve evaporar deixando as amostras padrão em um resíduo PEG-silano viscoso. Adicionar 20 mL de tolueno para o copo e mergulhar as amostras por 1-2 minutos. Remove as amostras do copo e enxaguar com tolueno, em seguida, isopropanol, e finalmente seco com nitrogênio. Neste ponto, as amostras devem olhar limpo ea camada de SAM deve ser invisível a olho nu. SAMs preparado desta maneira são utilizáveis ​​por até dois dias, quando armazenados em local seco (25 ° C, umidade moderada). Sem sucesso SAM resultados a formação de um revestimento de resíduo nublado ou irregular hued na superfície. </li></ol> Fabricar o counterelectrode Na mesma configuração de câmara de deposição utilizado para deposição de microeletrodos, faça uma counterelectrode depositando 1 nm de Cr, seguido por 4 nm de Au para um 22 x 50 mm N º 1 ou lamela de vidro n º 0. O counterelectrode deve ser quase transparente com um tom cinzento-claro ou rosa. Armazenar os counterelectrode revestido virado para cima em uma placa de Petri para mantê-lo limpo. Tubulina polimerizar em MTs <ol><li> Descongelar unlabeled tubulina e tubulina rodamina e imediatamente se combinam em uma proporção de 1:2 rotulados para unlabeled obter MTs brilhante. Mix pipetando cima e para baixo várias vezes. Polimerizar a tubulina para 20-40 minutos em um banho de água aquecida a 37 ° C. O comprimento do MTs será maior por mais tempo de polimerização. Enquanto a polimerização da tubulina é, prepare um tampão PIPES-taxol, adicionando taxol a 20 mM em um tubo de microcentrífuga contendo tampão PIPES. Misture o taxol bem pipetando cima e para baixo várias vezes e vórtex, e depois colocar o tubo no banho aquecida a 37 ° C. Após a polimerização, diluir a MTs por 1 / 100 em tampão PIPES-taxol quente e escudoda luz. MTs preparada desta forma alongada ao longo do tempo e pacote, que pode ser reduzido pela diluição. </li><li> Prepare uma mistura de oxigênio 10x de limpeza (OS) através da combinação de 30 mL tampão PIPES fria (0-4 ° C), 5 mL de 50% 2-mercaptoetanol, 5 mL de glicose oxidase, catalase 5 mL e 5 mL de glicose. 2 A glicose deve ser adicionado por último. Mix pipetando cima e para baixo 3-5 vezes após a adição de cada componente. Coloque a mistura OS no gelo, que permanecerá em vigor durante cerca de duas horas. </li><li> Verifique se a polimerização MT foi bem-sucedida e que o MTs são estáveis ​​sob vários minutos de excitação de fluorescência pela imagem da MTs preparado com 1x mix OS. Para geração de imagens a MTs preparado desta forma, não se esqueça de usar uma combinação de excitação / emissão de fluorescência filtro adequado para rodamina. Determinar a melhor diluição de trabalho para filamentos de imagens individuais por mais diluir a MTs por 1 / 10 a 1 / 100 com tampão PIPES e imagem com 1x mix OS. </li><li> Preparar uma câmara de fluxo, imprensando uma lamela de vidro de grandes dimensões (22 x 50 mm) e pequeno (22 x 22 mm) com dois pedaços de fita dupla-face colocada 2-3 mm. A fita é facilmente cortado em tiras, colocando-o sobre uma lâmina de vidro e corte com uma lâmina de barbear. Introduzir alguns microlitros de MTs na célula de fluxo, utilizando uma micropipeta. Para imagens de alta ampliação, os objectivos de longa distância de trabalho podem ser necessárias para geração de imagens da superfície superior da câmara de fluxo. A distância entre a superfície lamela / amostra até a superfície superior da câmara de fluxo é determinado pela espessura da fita e deve ser 60-100 mm. </li></ol> Padrão de MTs por eletroforese <ol><li> Prepare uma câmara de eletroforese, colocando duas tiras finas de fita dupla face em toda a amostra padrão, SAM-revestido de tal forma que o eletrodo está entre as tiras com o caminho de ligação perpendicular à fita (Fig. 2). Coloque o counterelectrode na fita dupla-face com o lado do metal tocar a fita de modo que a borda da amostra overhangs o counterelectrode por cerca de 3 mm (Fig. 2). Cortar vários comprimentos de 15 centímetros do fio de cobre isolados ímã e tira um centímetro do isolamento fora de ambas as extremidades. Anexar um fio para a área de Au exposta do substrato usando uma pequena gota de tinta prata tendo o cuidado de evitar fazer contato elétrico com o counterelectrode (isso pode ser verificado com um multímetro). Anexar um segundo fio à counterelectrode usando a pintura prata. Fios ligados com tinta de prata pode ser ainda mais segura para as amostras com fita. <img src="/files/ftp_upload/1390/1390fig2thumb.jpg" border="0" alt="figura 2" /> Figura 2. Montagem de células de fluxo para um microscópio invertido de fluorescência. Por favor, <a href="https://www.jove.com/files/ftp_upload/1390/1390fig2.jpg">clique aqui</a> para ver uma versão ampliada da figura 2. </li><li> Introduzir MTs na diluição desejada usando tampão PIPES combinado com OS em 1x de concentração final. Imagem com ampliação de 20x e localize o modelo sobre o wafer de silício antes da aplicação de uma voltagem. Opcionalmente, a câmara de fluxo pode ser selada com unha polonês para impedir fluxo de fluido e migração MT no plano da câmara de fluxo. </li><li> Aplicar uma tensão de até 1 V e observar a migração das MTs. Tensões mais baixas (até cerca de 0,5 V) pode ser usado se prendendo reversível de MTs é desejado, porém isto requer selando a câmara de fluxo para evitar desvio lateral. Unha polonês funciona bem para selar as extremidades abertas da câmara de fluxo. Em tensões acima de cerca de 0,7 V, cerca de adesão MT pode ser visto sobre o padrão, embora seja fraco. Em tensões acima de cerca de 0,9 V, a migração MT será mais rápido, ea adesão será mais forte. Acima de cerca de 1,2 V a probabilidade de indução de eletrólise (formação de bolhas de gás) e também para destruir o microeletrodos aumenta significativamente. A migração pode ser rastreado, ajustando o plano vertical do foco do microscópio. Se realizado corretamente, o MTs vai localizar na superfície do eletrodo. </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.

A migração dos MTs é facilmente visualizado através de microscopia de fluorescência por causa de seu brilho e da fluorescência de fundo baixa. O método é geralmente aplicável a padronização biomolécula, uma vez que só exige que as biomoléculas ser induzido a levar uma carga líquida pelo ajuste adequado do pH. Eletroosmose é evitada nesta configuração, porque não há superfícies carregadas como as paredes de sílica usada em eletroforese capilar. Várias modificações des...

No. 1 vidro borossilicato lamelas (22 X 50 mm) foram adquiridos da VWR. Polido de grade eletrônico do tipo p &lt;111&gt; wafers de silício foram adquiridos da Addison Engineering, Inc. alta pureza pintura prata foi obtida a partir Estrutura Probe, Inc. (Catalog # 5001 AB). Duplas multa bitola imã cobre (~ 0,08 milímetros (0,003) de diâmetro) podem ser obtidos a partir de empresas de abastecimento eletrônicos.

antifouling self-assembled monolayers on microelectrodes for patterning biomolecules

Nós apresentamos um procedimento para a formação de um poli (etileno glicol) (PEG) monocamada auto-montados trimetoxissilano (SAM) em um substrato de silício com microeletrodos de ouro. O PEG-SAM é formado em uma etapa única montagem e impede a incrustação em superfícies de silício e ouro. A SAM é usada para microeletrodos casaco estampados com a norma, litografia tom positivo. Usando o microtúbulos como um exemplo, nós aplicamos uma tensão DC para induzir a migração eletroforética para o eletrodo revestido SAM de forma reversível. A câmara de fluxo é usado para geração de imagens a migração eletroforética e padronização de microtúbulos<em&gt; In situ</em&gt; Meio de microscopia de epifluorescência. Este método é geralmente aplicável a padronização biomolécula, como se emprega a eletroforese para imobilizar moléculas-alvo e, portanto, não requer específicas interações moleculares. Além disso, evita problemas encontrados ao tentar padrão as moléculas SAM diretamente usando técnicas litográficas. A compatibilidade com litografia por feixe de elétrons permite que este método seja usado para biomoléculas padrão em 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

Nós apresentamos um procedimento para a formação de um poli (etileno glicol) monocamada auto-montados (PEG-SAM) em um substrato de silício com microeletrodos de ouro. O PEG-SAM é formada em uma única etapa e impede a incrustação em superfícies de silício e ouro. Eletroforese é então utilizado para padronização biomoléculas até a nanoescala.

Antifouling monocamadas auto-organizadas em Microeletrodos de Biomoléculas Patterning

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 visualizações.  Texas A&M University (TAMU). Nós apresentamos um procedimento para a formação de um poli (etileno glicol) monocamada auto-montados (PEG-SAM) em um substrato de silício com microeletrodos de ouro. O PEG-SAM é formada em uma única etapa e impede a incrustação em superfícies de silício e ouro. Eletroforese é então utilizado para padronização biomoléculas até a nanoescala.

Vídeo: 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.

Células padronização na opticamente transparentes Indium Tin Oxide Eletrodos

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

Biologia

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.

Padronização de Células-Tronco Embrionárias Usando o Chip Bio Virar

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 para medições única molécula de fluorescência de Superfície-imobilizada 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.

Profiling única célula transcricional dos cardiomiócitos de rato 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.

SABEDORIA Protein: A Workbench para<em&gt; In silico</em&gt;<em&gt; De novo</em&gt; Projeto 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.

Internalização e Observação de Fluorescent Biomoléculas em microorganismos vivos via Eletroporação

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.

Medição e Análise de Produção de Ácido Extracelular determinar Glicosidases Classificação

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.

O isolamento de regiões específicas genômico e identificação de moléculas pela Associated 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.

Quantificação de Estoicometria de Acetilação e Succinilação de Lisinas Proteicas em Sítios Específicos Usando Espectrometria de Massa de Aquisição de Dados Independente

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.

Ensaio de triagem à base de SA-β-galactosidase para a identificação de medicamentos Senoterapêuticos

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 ...