Este trabalho foi financiado pelo NIH / NIDCR Grant 1R01DE019796.

Os apresentados Leo-workflows permitem a rotulagem de isótopos estáveis ​​de proteína digere e peptídeos sintéticos. Estas experiências de cursos de tempo (Figura 1) são aplicáveis ​​a estudos comparativos e quantitativa do proteoma, bem como pesquisas protease. Cada fluxo de trabalho é constituído por duas etapas experimentais (figura 2): A) O tempo de amostragem do respectivo resolvido 18O-stable isotope reacção codificado (protease catalisada clivagem do péptido; protease catalisada reacção de permuta de oxigénio carboxilo; catalisada por ácido a troca de oxigénio carboxil reacção) e B), análise por espectrometria de massa e a representação gráfica de 18 S-cinética de incorporação. TimeCourse Experimentos A. I. paleo-TimeCourse: rotulagem Protease catalisada de clivagens proteolíticas <ol><li> (Opcional) ligações de dissulfureto de proteínas (10 ^ M) e os péptidos (250 nM) são reduzidas com DTT (concentração final 2,5 mM)em 25 mM NH 4 HCO 3 (ambos recentemente preparada) por incubação durante 30 min a 50 ° C. </li><li> (Opcional) cisteínas livres são alquiladas com iodoacetamida (concentração final 10 mM) em 25 mM NH 4 HCO 3 por incubação durante 30 min à temperatura ambiente no escuro. </li><li> Dependendo protease de interesse, proteína / peptídeo soluções precisam ser limpos-se para remover o tampão residual e agente de alquilação. Use colunas PepClean C-18 (Thermo rotação) para a limpeza do peptídeo e concentradores Vivaspin centrífuga (Sartorius) para troca de tampões para as amostras de proteína. </li><li> Redissolver / peptídeos / proteínas de câmbio em 20 ul de tampão de reacção da protease (ECE-1: 50 mM de MES-KOH, pH 5,5; tripsina: 25 mM NH 4 HCO 3, pH 8,0) contendo 1:1 (v / v) final H 2 18 O (95%, Sigma Isotec). </li><li> Retirar uma amostra de ponto de tempo zero, antes da adição da protease: Misturar 0,5 ul da mistura de reacção e 0,5 ul alfa ciano-4-hidroxicinâmicoácido matriz (10 mg / ml em 50% de acetonitrilo, 0,1% de TFA). Ponto de amostra numa placa de Opti-TOF MALDI alvo 384 (AB SCIEX) e deixar as gotas de solvente à temperatura ambiente até estar seco (cerca de 5 min). </li><li> Dividir a solução de reacção em duas aliquotas. A primeira alíquota será incubado com a protease de interesse (ECE-1: 75 nM; tripsina: 0,04 nM), à temperatura ambiente ou a temperatura recomendada para a enzima em particular. A segunda alíquota serão incubadas sem protease e servirá como amostra de controlo. A primeira alíquota da amostra representa um padrão para a protease catalisada 18 O-rotulagem e podem ser usados ​​para identificação de péptidos e proteínas, a detecção de actividades proteolíticas e monitorização das reacções de clivagem proteolitica. A segunda alíquota é utilizada para demonstrar que 18 O-incorporação é induzida pela protease e, portanto, nem a rotulagem nem clivagem é esperado para esta amostra. </li><li> Seguir a reacção por remoção de aliquotas da reacção e localizá-los como described no passo 5 em intervalos de tempo como parece em forma. As condições de reacção descritas acima são utilizados para monitorizar a reacção proteolítica por até quatro dias (aproximadamente 24 pontos). Iniciar a amostragem a cada 5 min até 30 min, em seguida, detectar a cada 30 minutos até 2 horas, depois a cada hora até 8 horas e a cada 8 horas até 4 dias. Produtos de clivagem deve aparecer dentro do primeiro 12 hr e do substrato deve ser completamente hidrolisada após 24 hr. Tempos de incubação prolongados de reacção permitem definir os produtos de reacção que são estáveis ​​discretos a partir de intermediários da reacção, os quais são posteriormente processados. Dependendo questão de pesquisa e dados enzima-substrato pares, manchar vezes e temperatura de incubação tem que ser modulada para assegurar amostragem reação ideal. </li><li> Após o ponto de tempo final de reacção é manchado ou em intervalos longos entre manchar, a placa alvo de MALDI é submetido a análise MALDI-TOF/TOF MS / MS como descrito a seguir (Secção B). </li></ol> II. Paleo-TimeCourse: Postdigrotulagem estion de terminais proteolíticas <ol><li> (Opcional) ligações de dissulfureto de proteínas (10 ^ M) e os péptidos (250 nM) são reduzidas com DTT (concentração final 2,5 mM) em 25 mM NH 4 HCO 3 (ambos preparados na hora) por incubação durante 30 min a 50 ° C. </li><li> (Opcional) cisteínas livres são alquiladas com iodoacetamida (concentração final 10 mM) em 25 mM NH 4 HCO 3 por incubação durante 30 min à temperatura ambiente no escuro. </li><li> Dependendo da protease de interesse, proteína / peptídeo soluções precisam de ser limpas-se para remover o tampão residual e do agente de alquilação. Use colunas PepClean C-18 de spin para limpeza de peptídeo e Vivaspin concentradores centrífugos para troca de tampão proteína. </li><li> Redissolver / trocar os produtos limpa-up peptídeos / proteínas em 20 ul de tampão de reacção (por exemplo, protease tripsina: 25 mM NH 4 HCO 3, pH 8,0) e digestão com protease de interesse para finalização (por exemplo, tripsina: 0,04 nM, 37 ° C, 12 hr). </li><li> Limpeza produtos de clivagem com colunas PepClean C-18 spin. Esta etapa irá eliminar atividades de protease residuais. </li><li> Redissolver / trocar os produtos limpa-up peptídicas em 20 ul de tampão de reacção da protease (tripsina: 25 mM NH 4 HCO 3, pH 8,0) contendo 1:1 (v / v) final H 2 O. 18 </li><li> Retirar uma amostra de ponto de tempo zero, antes da adição da enzima: mistura de 0,5 ul da mistura de reacção e 0,5 ul alfa matriz de ácido ciano-4-hidroxicinâmico (10 mg / ml em 50% de acetonitrilo, 0,1% de TFA). Ponto de amostra numa placa alvo de MALDI e deixar as gotas de solvente à temperatura ambiente até estar seco (cerca de 5 min). </li><li> Dividir a solução de reacção em duas aliquotas. A primeira alíquota será incubado com protease de interesse (por exemplo, tripsina: 0,04 nM), à temperatura ambiente ou a temperatura recomendada para a enzima em particular. A segunda alíquota serão incubadas sem protease e vai servir como um controlo. O primeiroalíquota representa uma amostra padrão de etiquetagem 18 protease catalisada postdigestion O-e pode ser utilizado para a quantificação de peptídeos e proteínas. A segunda alíquota é utilizada para demonstrar que 18 O-incorporação é catalisada pela protease e, portanto, não é esperado de rotulagem para esta amostra. </li><li> Seguir a reacção por remoção de aliquotas da reacção e localizá-los, tal como descrito no passo 7.) Em intervalos de tempo como parece adequada. Por exemplo, nós descoberto inicialmente a cada 5 min durante um período máximo de 30 minutos e a cada 15 minutos depois disso para monitorizar a reacção de permuta de oxigénio carboxil catalisada pela tripsina. </li><li> Após o ponto de tempo final de reacção é manchado ou em intervalos longos entre manchar a placa alvo de MALDI é submetido a análise MALDI-TOF/TOF MS / MS como descrito a seguir (Secção B). </li></ol> III. Aleo TimeCourse-: catalisada por ácido de rotulagem de grupos carboxilo <ol><li> Incubar péptidos individuais (50 nM) com 1:1 (v / v) de água 18 O-enriquecido em tele presença ou ausência (controlo) de 0,1% (v / v) de ácido trifluoroacético final (volume total 30 ul). </li><li> Os produtos de reacção de amostra diários durante 48 dias, por co-spotting uma alíquota de 0,5 ul da mistura de alfa 0.5μl com matriz de ácido ciano-4-hidroxicinâmico (10 mg / ml em 50% de acetonitrilo, 0,1% de TFA), directamente para um alvo MALDI placa. </li><li> Entre os intervalos de manchas e após avistar, do ponto final do tempo de reação apresentar MALDI placa-alvo para MALDI-TOF/TOF análise MS / MS, conforme descrito abaixo na Secção B. </li></ol> B. MALDI-TOF/TOF MS / MS de Aquisição de Dados e Análise <ol><li> Espectros de massa são adquiridos em um 4800 MALDI TOF / TOF Analyzer (AB SCIEX). </li><li> Antes da análise, o instrumento é calibrado com uma mistura de padrões de peptídeos (Kit de calibração para Padrões de Massa de AB SCIEX TOF / TOF instrumentos) com um máximo de tolerância do erro de medição de massa de ± 50 ppm e um número mínimo de seis picos para corresponder. </li><li> Os espectros de MS (intervalo de massas400 - 4000 m / z) são adquiridos em triplicado usando o modo de ião positivo com uma intensidade do laser ajustável (3400 - 3800; tamanho do passo 50), com uma gama aceitável a intensidade de base do pico de 2.000 - 45.000. Fotos individuais são adquiridos para espectros sub-, com 400 tiros total / espectro, parar condições entram em vigor depois de 800 sub-espectros são adquiridos (aprovação ou reprovação) ou 400 sub-espectros critérios de aceitação da passagem. No caso de amostras de baixa abundância ou na presença de complexos origens biológicas da extremidade inferior da gama de detecção MS deve ser aumentada para 800 m / z. Além disso, pode ser necessário para remover o sal e outros compostos que interferem com a etapa de limpeza de amostra, como descrito anteriormente, ou por separação por LC. </li><li> Arquivos de dados (MS. T2D arquivos) são exportados da Série Explorador 4000 software de aquisição de dados e importados para o nosso sistema de informações interno de laboratório, que utiliza software MASCOTE Distiller (Matrix Science) para processamento espectral e detecção de pico. Envelopes isotópicassão deconvoluted e 18 ó incorporação rácios automaticamente determinadas usando um algoritmo semelhante ao descrito por Mason et al. 12 e adaptado pelo nosso grupo de 9. Em alternativa, as ferramentas de software, tais como 13 e ZoomQuant Viper 14, bem como os pacotes de software comercial tais como BioWorks Xpress (Thermo Fisher Scientific) e Distiller Mascot Quantitation Toolbox (Matrix Science) pode deconvolute 18 O tipo de dados 15,16. 18 ó incorporação rácios são expressos como as contribuições relativas dos isótopos individuais espécies de péptidos (isto é, péptidos contendo 16 ó, 18 ou 18 O 1 O 2) a todo o envelope isotópica. </li><li> Para cada experiência TimeCourse, as massas moleculares ([M + H] +) para todas as espécies de péptidos detectados são extraídos a partir dos ficheiros de dados associados MS e os valores binned a uma largura de 100 ppm em massa. </li><li> AAo menos três [M + H] estão definidos para ser obrigado a preencher um escaninho. No caso de substratos conhecidos, a lista bin filtrado é comparado com uma lista de produtos de clivagem proteolítica previsto a partir da sequência de péptido de substrato utilizando a ferramenta FindPept ExPASy 17 ( <a href="http://au.expasy.org/tools/findpept.html" target="_blank">http://au.expasy.org/tools/findpept.html</a> ) e um 200 ppm em massa de tolerância de erro aceitação. </li><li> Espectros MS / MS são adquiridos para todos os valores de massa dos produtos de clivagem previstos pela ferramenta FindPept e para valores de bin que têm 18 O-incorporações que lhes estão associados. MS / MS de dados são adquiridos em 4800 MALDI Analyzer TOF / TOF em 1kV modo íon positivo refletor, com uma intensidade do laser fixa de 4200 e de gás-CID em modo desligado. 50 disparos são adquiridos num padrão aleatório por espectros de sub--se a um total de 40 sub-espectros por local (produzindo um total de 2.000 disparos / local). </li><li> MS / MS arquivos de dados (. T2D arquivos) são exportados a partir da série 4000 Explorer software de aquisição de dados e importados para o sistema de informação laboratorial em casa, os picos são detectados e listas de MS / MS dos picos estão associados com os dados correspondentes binned MS. </li><li> Para identificação peptídeo, listas de MS / MS de pico são pesquisados ​​no banco de dados SwissProt usando o motor de busca MASCOTE com os parâmetros de pesquisa a seguir: não especificidade enzima, 150 ppm e íon precursor 0,2 fragmento Da tolerâncias de massa de íons. </li><li> Identificações peptídeos podem adicionalmente ser validado com o Data Explorer software (AB SCIEX), confirmando as características 18 O incorporação padrões de íons através y série de fragmentos, como descrito por Shevchenko et al. 18. </li></ol> C. Preparação do Tempo espectral e 18 Ó incorporação de parcelas Parcelas de tempo espectrais: arquivos de dados (MS. T2D arquivos) para cada ponto de tempo de reação são exportados do software Data Explorer como ASCII-arquivos usando ummacro e importados para um programa de análise de dados e software gráfico (por exemplo de origem por OriginLab) e exibido como parcelas de cascata (Figura 3). 18 O-incorporação parcelas: Para cada um dos produtos de clivagem do péptido binned, as contribuições relativas das espécies de isótopos de péptidos individuais (16 O, 18 O 18 O 1 ou 2) são extraídos através de todos os pontos de tempo de reacção e representada graficamente em função do tempo (Figura 4).

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Através da combinação de rotulagem isótopo estável e de elevada resolução em espectrometria de massa de uma forma resolvida no tempo, o método Paleo TimeCourse-permite uma análise dinâmica da geração de produtos peptídicos. O ensaio pode ser utilizado para gerar péptidos marcados isotopicamente estáveis ​​para estudos quantitativos e qualitativos proteómica e avaliar a cinética através da qual os péptidos proteotypic são gerados. Além disso, Paleo TimeCourse foi concebido para avaliar as vias pr...

<table border="1"><tbody><tr><td> Nome de material </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Colunas PepClean C-18 de Spin </td><td> Thermo </td><td> 89870 </td></tr><tr><td> Opti-TOF 384 placa-alvo MALDI </td><td> AB SCIEX </td><td> 1016629 </td></tr><tr><td> 4800 MALDI TOF / TOF </td><td> AB SCIEX </td><td></td></tr></tbody></table> Tabela 1. Materials <table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> O ácido alfa-ciano-4-hidroxicinâmico </td><td> Sigma Aldrich </td><td> 70990-1G-F </td></tr><tr><td> Albumina de soro bovino (BSA) </td><td> Sigma Aldrich </td><td> A3294-10G </td></tr><tr><td> Ditiotreitol (DTT) </td><td&gt; Acros </td><td> 16568-0050 </td></tr><tr><td> Iodoacetamida (IAM) </td><td> Sigma Aldrich </td><td> 1149-5G </td></tr><tr><td> Enzima conversora de endotelina-1 (ECE-1) </td><td> R &amp; D Systems </td><td> 1784-ZN </td></tr><tr><td> Tripsina Ouro </td><td> Promega </td><td> V5280 </td></tr><tr><td> Água-18 O, 97% átomo de 18 O </td><td> Sigma Aldrich </td><td> 329878-1G </td></tr><tr><td> Ácido trifluoroacético (TFA) </td><td> Thermo </td><td> 28904 </td></tr><tr><td> Kit Padrões de massa para Calibração de AB SCIEX TOF / TOF instrumentos </td><td> AB SCIEX </td><td> 4333604 </td></tr></tbody></table> Tabela 2. Reagentes

protease- and acid-catalyzed labeling workflows employing 18o-enriched water

Isótopos estáveis ​​são ferramentas essenciais em espectrometria de massa biológica. Historicamente, 18 O-isótopos estáveis ​​têm sido extensivamente utilizadas para estudar os mecanismos catalíticos das enzimas proteolíticas 1-3. Com o advento da espectrometria de massa à base de proteomics, a incorporação catalisada enzimaticamente, de 18 átomos de O-água isotopicamente enriquecido estável tornou-se um método popular para comparar quantitativamente os níveis de proteína de expressão (revisto por Fenselau e Yao 4, Miyagi e Rao 5 e Ye et al. 6). 18 O-rotulagem constitui uma alternativa simples e de baixo custo para produtos químicos (por exemplo iTRAQ, ICAT) e metabólicas (por exemplo, técnicas de SILAC) rotulagem 7. Dependendo da protease utilizada, 18 O-rotulagem podem resultar na incorporação de até dois átomos de O-18 no grupo carboxilo C-terminal do produto de clivagem 3 </sup&gt;. A reacção de marcação pode ser subdividido em dois processos independentes, a clivagem da ligação peptídica e a reacção de permuta de oxigénio carboxil 8. Em nossa Paleo (p rotease-a e Abeling ssisted l mploying 18 O enriquecido água) adaptação de enzimática 18 O rotulagem, utilizamos 50% de água 18 O enriquecido para produzir assinaturas isotópicas distintas. Em combinação com a alta resolução de laser assistida por matriz de ionização de dessorção de tempo-de-voo espectrometria de massa tandem (MALDI-TOF/TOF MS / MS), os envelopes isotópicos característicos podem ser usados ​​para identificar os produtos de clivagem com um elevado grau de especificidade. Nós já ter usado o Paleo metodologia para detectar e caracterizar proteases endógenas 9 e monitorar reações proteolíticas 10-11. Desde Paleo codifica a própria essência da reação de clivagem proteolítica, a instalação experimental é Enri simples e bioquímicoschment passos de produtos de clivagem podem ser contornados. A paleo-método pode ser facilmente estendido para (i) o curso experiências que monitoram a dinâmica de reações de clivagem proteolítica e (ii) a análise da proteólise em amostras biológicas complexas que representam condições fisiológicas. Paleo-TimeCourse experimentos ajudar a identificar passos limitação de taxa de processamento e intermediários de reação em complexas reações via proteolíticas. Além disso, a reacção de Paleo permite-nos identificar as enzimas proteolíticas como a tripsina serina-protease que é capaz de associar novamente os seus produtos de clivagem e de catalisar a incorporação de um 18 segundo O-átomo. Tais &quot;etiquetagem dupla&quot; enzimas podem ser utilizados para postdigestion 18 O-rotulagem, em que os péptidos marcados são exclusivamente através da reacção de oxigénio carboxil troca. A nossa estratégia terceira estende rotulagem empregando água 18 ó enriquecido além enzimas e utiliza condições de pH ácido para introduzir 18 sinal isótopo estável O-Atures em peptídeos.

Utilizou-se o fluxo de trabalho Paleo TimeCourse para monitorar dinamicamente a incorporação de 18 O-isótopos estáveis ​​em produtos de clivagem de péptidos geradas por enzimas proteolíticas. A abordagem apresentada é uma ferramenta versátil para estudar comparativamente vias de processamento proteolíticas de diferentes substratos e combinações de protease. Por amostragem reacções proteolíticas repetidamente durante o curso da reacção, o experimento Paleo TimeCourse fornece instantâneos r...

Watch this Scientific Journal Video about Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water at JoVE.com

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Fluxos de trabalho de isótopos estáveis ​​de rotulagem empregando<sup&gt; 18</sup&gt; O-água enriquecida (Leo-fluxos de trabalho) são ferramentas versáteis para estudos quantitativos e qualitativos proteômica. Nos fluxos de trabalho protease assistidas (Paleo),<sup&gt; 18</sup&gt; O-átomos são introduzidos por clivagem proteolítica e reacções de permuta de oxigénio carboxilo mediada por proteases. No catalisada por ácido de fluxo de trabalho (Aleo),<sup&gt; 18</sup&gt; O-átomos são introduzidos por troca de oxigénio carboxilo a pH baixo.

Protease e catalisada por ácido Workflows Labeling Empregando<sup&gt; 18</sup&gt; O-enriquecido Água

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic ...

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JoVE Journal

Biology

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

11.9K visualizações.  Boston Biomedical Research Institute. Fluxos de trabalho de isótopos estáveis ​​de rotulagem empregando 18 O-água enriquecida (Leo-fluxos de trabalho) são ferramentas versáteis para estudos quantitativos e qualitativos proteômica. Nos fluxos de trabalho protease assistidas (Paleo), 18 O-átomos são introduzidos por clivagem proteolítica e reacções de permuta de oxigénio carboxilo mediada por proteases. No catalisada por ácido de fluxo de trabalho (Aleo),

Vídeo: Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release.  FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated.  Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence.  Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.



The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

Medição Exocitose em Neurônios Usando Labeling FM

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

Biologia

Morris Water Maze Experiment

The Morris water maze is widely used to study spatial memory and learning. Animals are placed in a pool of water that is colored opaque with powdered non-fat milk or non-toxic tempera paint, where they must swim to a hidden escape platform. Because they are in opaque water, the animals cannot see the platform, and cannot rely on scent to find the escape route. Instead, they must rely on external/extra-maze cues. As the animals become more familiar with the task, they are able to find the platform more quickly. Developed by Richard G. Morris in 1984, this paradigm has become one of the "gold standards" of behavioral neuroscience.

The Morris water maze is a well-accepted tool used to document the involvement of the hippocampus in a behavioral task.

Morris Experiment Maze Água

The Morris water maze is widely used to study spatial memory and learning. Animals are placed in a pool of water that is colored opaque with powdered ...

Sigma's Non-specific Protease Activity Assay - Casein as a Substrate

Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases. Sigma's non-specific protease activity assay may be used as a standardized procedure to determine the activity of proteases, which is what we do during our quality control procedures. In this assay, casein acts as a substrate. When the protease we are testing digests casein, the amino acid tyrosine is liberated along with other amino acids and peptide fragments. Folin and Ciocalteus Phenol, or Folin's reagent primarily reacts with free tyrosine to produce a blue colored chromophore, which is quantifiable and measured as an absorbance value on the spectrophotometer. The more tyrosine that is released from casein, the more the chromophores are generated and the stronger the activity of the protease. Absorbance values generated by the activity of the protease are compared to a standard curve, which is generated by reacting known quantities of tyrosine with the F-C reagent to correlate changes in absorbance with the amount of tyrosine in micromoles. From the standard curve the activity of protease samples can be determined in terms of Units, which is the amount in micromoles of tyrosine equivalents released from casein per minute.
To view this article in Chinese, <a href="http://jove.com/index/details.stp?ID=910">click here</a>

Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases. Sigma's non-specific protease activity assay may be used as a standardized procedure to determine the activity of proteases.

Não-específicas da Sigma ensaio de atividade de protease - A caseína como substrato

Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases.  Sigma's non-specific protease ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

Labeling fluorescentes de Drosophila Estruturas Coração

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

Uso do Kit de Detecção de Protease Fluorescente para determinar a atividade de protease

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae.  In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies.  The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins.  Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

Preparação de Abóboras Drosophila cromossomos politênicos para Rotulagem de anticorpos

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological ...

ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells. While fluorescent 
proteins such as green fluorescence protein (GFP) have been extensively used as fusion partners to proteins to track the properties of a protein of interest1, recent 
developments with smaller tags enable new functionalities of proteins to be examined in cells such as conformational change and protein-association 2, 3. One small 
tag system involves a tetracysteine motif (CCXXCC) genetically inserted into a target protein, which binds to biarsenical dyes, ReAsH (red fluorescent) and FlAsH 
(green fluorescent), with high specificity even in live cells 2. The TC/biarsenical dye system offers far less steric constraints to the host protein than 
fluorescent proteins which has enabled several new approaches to measure conformational change and protein-protein interactions 4-7. We recently developed 
a novel application of TC tags as sensors of oligomerization in cells expressing mutant huntingtin, which when mutated aggregates in neurons in Huntington 
disease 7. Huntingtin was tagged with two fluorescent dyes, one a fluorescent protein to track protein location, and the second a TC tag which only 
binds biarsenical dyes in monomers. Hence, changes in colocalization between protein and biarsenical dye reactivity enabled submicroscopic oligomer 
content to be spatially mapped within cells. Here, we describe how to label TC-tagged proteins fused to a fluorescent protein (Cherry, GFP or CFP) 
with FlAsH or ReAsH in live mammalian cells and how to quantify the two color fluorescence (Cherry/FlAsH, CFP/FlAsH or GFP/ReAsH combinations).

The biarsenical dyes FlAsH and ReAsH bind specifically to tetracysteine motifs in proteins and can selectively label proteins in live cells. Recently this labeling strategy has been used to develop sensors for different protein conformations or oligomeric states. We describe the labeling approach and methods to quantitatively analyze binding.

ReAsH / Flash Rotulagem e Análise de Imagem Sensor de Proteínas em Células Tetracysteine

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells.  While fluorescent 
proteins ...

GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. Current strategies for microbial analysis include both traditional laboratory culture-dependent techniques and those based on direct extraction and determination of certain biomarkers1, 2. Few among the diversity of microbial species inhabiting soil can be cultured, so culture-dependent methods introduce significant biases, a limitation absent in biomarker analysis.
The glucosamine, mannosamine, galactosamine and muramic acid have been well served as measures of both the living and dead microbial mass, of these the glucosamine (most abundant) and muramic acid (uniquely from bacterial cell) are most important constituents in the soil systems3, 4. However, the lack of knowledge on the analysis restricts the wide popularization among scientific peers. Among all existing analytical methods, derivatization to aldononitrile acetates followed by GC-based analysis has emerged as a good option with respect to optimally balancing precision, sensitivity, simplicity, good chromatographic separation, and stability upon sample storage5.
Here, we present a detailed protocol for a reliable and relatively simple analysis of glucosamine and muramic acid from soil after their conversion to aldononitrile acetates. The protocol mainly comprises four steps: acid digestion, sample purification, derivatization and GC determination. The step-by-step procedure is modified according to former publications6, 7. In addition, we present a strategy to structurally validate the molecular ion of the derivative and its ion fragments formed upon electron ionization. We applied GC-EI-MS-SIM, LC-ESI-TOF-MS and isotopically labeled reagents to determine the molecular weight of aldononitrile acetate derivatized glucosamine and muramic acid; we used the mass shift of isotope-labeled derivatives in the ion spectrum to investigate ion fragments of each derivatives8. In addition to the theoretical elucidation, the validation of molecular ion of the derivative and its ion fragments will be useful to researchers using &delta;13C or ion fragments of these biomarkers in biogeochemical studies9, 10.

We describe a method protocol for the GC-based analysis of the aldonitrile acetate derivatives of glucosamine and muramic acid extracted from soil. For elucidation of the chemical mechanism, we also present a strategy to confirm the structure of the derivative and the ion fragments formed upon electron ionization.

GC baseado em detecção de ácido Aldononitrile Glucosamina e Murâmicos Acetato derivatizados à determinação do resíduo microbiana em solo

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

Medindo a progressão do ciclo celular Cinética com marcação metabólica e Citometria de Fluxo

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

Rato Ilhéu de Langerhans isolamento usando uma combinação de colagenase purificada e Protease Neutra

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...