Este trabalho foi financiado pelos Institutos Canadenses de Pesquisa em Saúde (CIHR), Nacional de Ciência e Engenharia Research Council (NSERC), e um treinamento CIHR bolsa de estudo (para LF). Agradecemos ao Prof Gerard D. Wright (McMaster University, Canadá) para a AAC (6)-Ii expressão plasmídeo.

1. Preparar soluções estoque <ol><li> Purificar a macromolécula de interesse. (Neste caso, aminoglicosídeo N-6&#39;-acetiltransferase-Ii (AAC6&#39;-II), é isolada como relatado em outros lugares. 13) </li><li> Prepare 4 litros de solução de diálise. (Neste caso foi utilizado 25 mM 4 - (2-hidroxietil)-1-piperazineethanesulfonic ácido (HEPES, 238,3 MW g / mol), contendo 2 mM de ácido etilenodiaminotetracético (EDTA, 292,2 MW), no pH 7,5). </li><li> Diálise a proteína A AAC amostra (6 &#39;)-II (5 mL a 400 mM) utilizados devem ser dialisada em tampão de diálise (3 x 1.3 L). A solução de diálise final é mantida para lavar a máquina e para diluir amostras. </li><li> Filtrar a solução de diálise final através de um filtro de celulose 0,45 mM, e armazenados a 4 ° C. Será referido como &quot;tampão de corrida&quot; </li><li> Solução de proteína de filtro através de um filtro de seringa 0,2 m que tenha sido lavado cuidadosamente com tampão de corrida. Medir a concentração final da proteína através de um ensaio padrão (Bradford, Lowry, etc) ou absorbância UV. Loja da proteína para maximizar a estabilidade a longo prazo. (No caso da AAC (6 &#39;)-Ii, isto significa que o armazenamento a 4 ° C. AAC (6&#39;)-Ii não mantém atividade após congelamento e descongelamento). </li><li> Prepare 200μL de 25 mM solução estoque de acetil coenzima A (AcCoA, o ligante, MW 809,57) dissolvendo 4,0 mg na execução de buffer. Congelar a -78 ° C até que esteja pronto para uso. </li><li> Todas as proteínas e as amostras de ligante devem ser originários da mesma ação de soluções, para minimizar amostra aleatória com o modelo flutuações na concentração. Um fator de correção único para a concentração de proteína em toda a variável c dataset é ajustado na análise. 10 </li></ol> 2. Preparação de amostras ITC <ol><li> Diluir a solução de enzima a um c-valor de 64 com um volume final de 2 mL. (No caso da AAC (6 &#39;)-Ii, isto corresponde a 192 mM). </li><li> Descongelar rapidamente o ligante solução-mãe (AcCoA) em água gelada. </li><li> Prepare 0,5 mL de uma solução AcCoA 10 vezes mais concentrada, em seguida, o número de sítios de ligação na proteína, neste caso 4 mM, diluindo-se 80 mL da solução estoque em 420 mL AcCoA de tampão de corrida. Transferir para um tubo de enchimento da pipeta. Retornar rapidamente a solução estoque AcCoA a -78 ° C. </li><li> Degas proteína e soluções sob vácuo por 5 min a uma temperatura de 1 ° C abaixo da temperatura desejada em execução (19 ° C). </li></ol> 3. Configurando a seringa 14 <ol><li> Inserir a seringa de injeção através de titular seringa até a braçadeira seringa pré-montada é na mesma altura que o titular. </li><li> Alimentar o clamp seringa, o segundo sobre a seringa de injeção até que esteja firmemente pressionado contra a parte inferior do titular seringa. Aperte levemente o grampo com o previsto &quot;Drive Bola 0,050 Hex Point. </li><li> Coloque titular seringa no suporte da pipeta. </li><li> Deslize suavemente o injector pipeta dentro da seringa de injeção. Verifique se a ponta do êmbolo é alimentado diretamente para o buraco da seringa. Depois de completamente inserido, o anel de travamento de rosca do titular da seringa no injector da pipeta. </li></ol> 4. Carregando a célula de amostra 14 <ol><li> Lavar célula de amostra com um mínimo de 50 mL de tampão de corrida, e remover qualquer líquido restante usando uma longa agulha 2,5 ml seringa de vidro. </li><li> Lentamente desenhar um mínimo de 1,8 ml de solução de proteína na amostra limpa e seca seringa ml de longa agulha 2.5. Tenha cuidado para não introduzir quaisquer bolhas. </li><li> Cuidadosamente inserir a agulha em célula de amostra e gentilmente tocar o fundo da célula. Levantar a ponta ligeiramente (~ 1 mm) e gentilmente injetar AAC (6 &#39;)-solução Ii dentro da célula até o excesso de líquido é visível acima da parte superior da célula de amostra. </li><li> Lentamente levantar a agulha cerca de 1 cm, garantindo permanece líquido no overflow. Rapidamente retirar e injetar uma pequena quantidade de solução (~ 0,25 ml) para remover quaisquer bolhas de presos na cela da amostra. </li><li> Remover todos estouro solução. Isto é conseguido, deslizando suavemente a agulha ao lado de transbordamento para dentro da célula da amostra. A ponta da seringa vai bater uma borda, esta é a altura desejo para a solução em execução. Retire todo o líquido que fica acima desta linha. </li></ol> 5. Carregando seringa de injeção e iniciar executar 14 <ol><li> Conecte a seringa de plástico do tubo de carga no porto de preenchimento. </li><li> Ponta inferior êmbolo para a parte superior da porta de preenchimento. </li><li> Coloque a solução AcCoA no tubo de enchimento da pipeta na parte inferior do titular da pipeta. A seringa não deve tocar o fundo do tubo de enchimento. </li><li> Tirar lentamente a solução para a seringa até que uma pequena quantidade entra no tubo da seringa carregamento. </li><li> Feche a porta encher, diminuindo a ponta do êmbolo e clique no botão Fechar porta Fill. Purga e recarga, clicando no botão Limpar-&gt; Refill, 3 vezes para remover quaisquer bolhas de ar que pode ter ficado preso durante o carregamento. </li><li> Remova o tubo de enchimento do detentor pipeta e limpe a ponta da seringa. </li><li> Tome pipeta montagem e seringa suavemente menor para dentro da célula da amostra. Prossiga lentamente como a seringa pode facilmente dobrar, por isso muito cuidado é necessário. Garantir a seringa está completamente inserido, pressionando para baixo na base de anel externo. </li><li> Definir a temperatura desejada em execução (neste caso 20 ° C), e selecione uma potência de referência que é ligeiramente maior do que a vazão máxima de injeção de calor esperado (neste caso 20μcal/sec). </li><li> O programa de injeção de volumes desejados e atrasos. (Neste caso, 28 injeções foram empregados. A primeira injeção teve um volume de 2 mL com um atraso s 60. Todas as injeções subseqüentes tiveram volumes de 10 mL com 330 atrasos s). </li></ol> 6. Executa subseqüentes <ol><li> Em todas as execuções subseqüentes repita os passos 2-5, enquanto diminui AAC (6 &#39;)-II e concentrações AcCoA por um fator de 2,4,8,16 e 32. </li></ol> 7. Análise de Dados <ol><li> Use um procedimento de ajuste, que globalmente se encaixa todas as isotermas de um único conjunto de parâmetros de ligação, como descrito anteriormente 10. </li></ol> 8. Resultados representante Dados representativos são mostrados na Figura 1. As formas das isotermas deve variar com a concentração. Transições mais nítidas são esperados para maior c-valores (ou seja, com elevado teor proteico e as concentrações de ligante) (Figura 2). No caso da AAC (6 &#39;)-II, o modelo de dois-site seqüencial dá um melhor ajuste do que um descrevendo dois conjuntos de idênticas, sites independentes, com estequiometrias ajustável. <img src="/files/ftp_upload/2529/2529fig1.jpg" alt="Figura 1" /> Figura 1. Isotermas produzido pela titulação de AcCoA (3,86 mM) na AAC (6 &#39;)-II (192 mm). A) trace ITC Raw. B) Os valores integrado utilizado para determinar os parâmetros de ligação (quadrados) com um encaixe 2 sites seqüencial (-). <img src="/files/ftp_upload/2529/2529fig2.jpg" alt="Figura 2" /> Isotermas figura 2. ITC para AcCoA titulada em AAC (6 &#39;)-II em concentrações variadas. Os dados experimentais (círculos abertos) estavam aptos para um modelo de 2 sets-de-sites independentes (magenta tracejada) e um modelo 2-site seqüencial (azul sólidos). O modelo 2-site sequentional claramente dá acordo de melhor no geral. As concentrações utilizadas foram A) 6 mM, 0,25 mM, B) 12 mM, 0,25 mM, C) 24 mM, 0,5 mM, D) 48 mM, 1,0 mM, E) 96 mM, 1,9 mM, e F) 196 mM, 3,86 mM, para AAC (6 &#39;)-II e AcCoA respectivamente.

<ol>
	<li>Cliff, M. J., Ladbury, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+year+2002+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2002 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 16, 383-391 (2003).</li><li>Cliff, M. J., Gutierrez, A., Ladbury, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+year+2003+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2003 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 17, 513-523 (2004).</li><li>Ababou, A., Ladbury, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survey+of+the+year+2004:+literature+on+applications+of+isothermal+titration+calorimetry.">Survey of the year 2004: literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 19, 79-89 (2006).</li><li>Ababou, A., Ladbury, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survey+of+the+year+2005:+literature+on+applications+of+isothermal+titration+calorimetry.">Survey of the year 2005: literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 20, 4-14 (2007).</li><li>Okhrimenko, O. k. s. a. n. a., J, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+year+2006+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2006 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 21, 1-19 (2008).</li><li>Bjelic, S., Jelesarov, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+year+2007+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2007 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 21, 289-312 (2008).</li><li>Leavitt, S., Freire, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+protein+binding+energetics+by+isothermal+titration+calorimetry.">Direct measurement of protein binding energetics by isothermal titration calorimetry.</a> Current Opinion in Structural Biology. 11, 560-566 (2001).</li><li>Wiseman, T., Williston, S., Brandts, J. F., Lin, L. -. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+measurement+of+binding+constants+and+heats+of+binding+using+a+new+titration+calorimeter.">Rapid measurement of binding constants and heats of binding using a new titration calorimeter.</a> Analytical Biochemistry. 179, 131-137 (1989).</li><li>Capaldi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+X-Ray+Structure+of+Zebrafish+(Danio+rerio)+Ileal+Bile+Acid-Binding+Protein+Reveals+the+Presence+of+Binding+Sites+on+the+Surface+of+the+Protein+Molecule.">The X-Ray Structure of Zebrafish (Danio rerio) Ileal Bile Acid-Binding Protein Reveals the Presence of Binding Sites on the Surface of the Protein Molecule.</a> Journal of Molecular Biology. 385, 99-116 (2009).</li><li>Freiburger, L. A., Auclair, K., Mittermaier, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidating+Protein+Binding+Mechanisms+by+Variable-c+ITC.">Elucidating Protein Binding Mechanisms by Variable-c ITC.</a> ChemBioChem. 10, 2871-2873 (2009).</li><li>Wybenga-Groot, L. E., Draker, K. -. a., Wright, G. D., Berghuis, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+an+aminoglycoside+6'-N-acetyltransferase:+defining+the+GCN5-related+N-acetyltransferase+superfamily+fold.">Crystal structure of an aminoglycoside 6'-N-acetyltransferase: defining the GCN5-related N-acetyltransferase superfamily fold.</a> Structure. 7, 497-507 (1999).</li><li>Draker, K., Northrop, D. B., Wright, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+Mechanism+of+the+GCN5-Related+Chromosomal+Aminoglycoside+Acetyltransferase+AAC(6')-Ii+from+Enterococcus+faecium:+Evidence+of+Dimer+Subunit+Cooperativity.">Kinetic Mechanism of the GCN5-Related Chromosomal Aminoglycoside Acetyltransferase AAC(6')-Ii from Enterococcus faecium: Evidence of Dimer Subunit Cooperativity.</a> Biochemistry. 42, 6565-6574 (2003).</li><li>Wright, G. D., Ladak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+and+characterization+of+the+chromosomal+aminoglycoside+6'-N-acetyltransferase+from+Enterococcus+faecium.">Overexpression and characterization of the chromosomal aminoglycoside 6'-N-acetyltransferase from Enterococcus faecium.</a> Antimicrob. Agents Chemother. 41, 956-960 (1997).</li><li>MicroCal. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ITC Data Analysis in Origin. , (2004).</li></ol>

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Esta parte analítica da variável c montagem foi descrito anteriormente em detalhe 10. Aqui relatamos os aspectos práticos da coleta variável c conjuntos de dados adequados para esta abordagem. É essencial que todas as proteínas e as amostras de ligante são desenhados a partir das soluções estoque mesmo. Portanto, é importante que a solução estoque suficiente é preparado inicialmente para completar toda a série de experimentos. Isso garante que o rácio de AAC (6 &#39;)-II e AcCoA é constante en...

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		<div>
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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Acetyl coenzyme A (AcCoA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A2056</td>
<td>&nbsp;</td>
<tr>
<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>7365-45-9</td>
<td>&nbsp;</td>
<tr>
<td>ethylenediaminetetraacetic acid (EDTA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>431788</td>
<td>&nbsp;</td>
<tr>
<td>Spectra/Por 2 Dialysis Tubing</td>
<td>&nbsp;</td>
<td>Spectrum Labs</td>
<td>132678</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Syringe Filter (0.2 &mu;m)</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>281445-477</td>
<td>&nbsp;</td>
<tr>
<td>Cellulos Nitrate Membrane Filters (0.45 &mu;m)</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>7184-004</td>
<td>&nbsp;</td>
<tr>
<td>VP-ITC</td>
<td>&nbsp;</td>
<td>MicroCal</td>
<td>VP-ITC</td>
<td>Microcalorimeter used for measurements</td>
</tr>
<tr>
<td>ThermoVac</td>
<td>&nbsp;</td>
<td>MicroCal</td>
<td>USB Thermo Vac</td>
<td>Temperature Controlled Degassing Station</td>
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collecting variable-concentration isothermal titration calorimetry datasets in order to determine binding mechanisms

Calorimetria de titulação isotérmica (ITC) é comumente usada para determinar os parâmetros termodinâmicos associados com a ligação de um ligante para uma macromolécula host. ITC tem algumas vantagens sobre abordagens comuns espectroscópicas para o estudo de host / ligante interações. Por exemplo, o calor liberado ou absorvido quando os dois componentes interagem é medido diretamente e não requer qualquer repórteres exógena. Assim, a entalpia de ligação ea constante de associação (Ka) são obtidos diretamente a partir de dados ITC, e pode ser usado para calcular a contribuição entrópica. Além disso, a forma da isoterma é dependente do c-valor e do modelo mecanicista envolvidos. O c-valor é definido como n = c [P] TKA, onde [P] t é a concentração de proteína, e n é o número de sites de ligação do ligando dentro do hospedeiro. Em muitos casos, vários sítios de ligação para um ligante dadas não são equivalentes e ITC permite a caracterização dos parâmetros termodinâmicos de ligação para cada site individual vinculativo. Este, porém, exige que o modelo correto de ligação ser usado. Essa escolha pode ser problemático se os diferentes modelos podem caber os mesmos dados experimentais. Nós temos mostrado previamente que este problema pode ser contornado através da realização de experimentos em vários c-valores. As isotermas múltiplos obtidos em diferentes valores de c estão aptos simultaneamente a modelos distintos. O modelo correto é o próximo identificados com base na bondade de ajuste em todo o conjunto de dados variável c-inteiro. Este processo é aplicado aqui para o aminoglicosídeo resistência causando aminoglicosídeo enzima N-6&#39;-acetiltransferase-Ii (AAC (6 &#39;)-II). Embora nossa metodologia é aplicável a qualquer sistema, a necessidade dessa estratégia é melhor demonstrada com um sistema de macromolécula-ligante mostrando allostery ou cooperatividade, e quando diferentes modelos de ligação fornecer cabe essencialmente idênticos aos mesmos dados. Ao nosso conhecimento, não existem tais sistemas disponíveis comercialmente. AAC (6 &#39;)-II, é um homo-dímero contendo dois sítios ativos, mostrando cooperatividade entre as duas subunidades. Contudo, os dados obtidos ITC em um único c valor pode ser apto igualmente bem a pelo menos dois modelos diferentes de um modelo de dois conjuntos-de-sites independentes e um dois-site seqüencial modelo (cooperativa). Através da variação da c-valor, como explicado acima, estabeleceu-se que o modelo correto de ligação para AAC (6 &#39;)-II é um dois-site modelo seqüencial de ligação. Aqui, descrevemos os passos que devem ser tomados ao realizar experimentos ITC para obter conjuntos de dados adequados para a variável-c análises.

Watch this Scientific Journal Video about Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms at JoVE.com

Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

ITC é uma ferramenta poderosa para estudar a ligação de um ligante para seu hospedeiro. Em sistemas complexos no entanto, vários modelos podem ajustar os dados igualmente bem. O método descrito aqui fornece um meio para elucidar o modelo de encadernação adequada para sistemas complexos e extrair os parâmetros termodinâmicos correspondentes.

Coleta variável concentração Datasets Calorimetria Isotérmica de Titulação a fim de determinar mecanismos de ligação

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host ...

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Biology

12.7K visualizações.  McGill University. ITC é uma ferramenta poderosa para estudar a ligação de um ligante para seu hospedeiro. Em sistemas complexos no entanto, vários modelos podem ajustar os dados igualmente bem. O método descrito aqui fornece um meio para elucidar o modelo de encadernação adequada para sistemas complexos e extrair os parâmetros termodinâmicos correspondentes.

Vídeo: Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

Titration of Human Coronaviruses Using an Immunoperoxidase Assay

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for prototype strains 229E and OC43 of human coronavirus (HCoV). Therefore, an alternative indirect immunoperoxidase assay (IPA) was developed for the detection and titration of these viruses and is described herein. Susceptible cells are inoculated with serial logarithmic dilutions of virus-containing samples in a 96-well plate format. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as 'Tissue Culture Infectious Dose 50 percent' (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain infectious replicating virus. This technique provides a reliable method for the titration of HCoV-229E and HCoV-OC43 in biological samples such as cells, tissues and fluids. This article is based on work first reported in Methods in Molecular Biology (2008) volume 454, pages 93-102.

In this video, we demonstrate an alternative method for detection and titering of viruses using an enzymatic antigen detection technique known as an immunoperoxidase assay. Here, we will show you how to collect your viral samples, prepare the cells for testing, and finally the immunoperoxidase assay using serial dilutions to determine the viral titer.

Titulação de coronavírus humanos Usando um ensaio de imunoperoxidase

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for ...

Biologia

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds 

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. Collecting site-specific biochemical markers allows us to evaluate the relationship between surgical wound and serum levels; determine any associations between mediator release, pain and analgesic consumption; and evaluate the effect of systemic and peripheral drug administration on surgical wound biochemistry.
This methodology has been applied to healthy women undergoing elective cesarean delivery with spinal anesthesia. Wound exudate and serum mediators, in conjunction with pain scores and analgesics consumption were measured at 1, 6, 24, and 48 hours post-cesarean delivery. Biochemical mediators that were detected included IL-1&beta;, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNF&alpha;, INF&gamma;, G-CSF, GM-CSF, MCP-1 and MIP-1&beta;, nerve growth factor (NGF), prostaglandin E2 (PG-E2) and substance P. We found no correlations between wound and serum cytokines concentrations or time-release profiles (J Pain. 2008 Jul 9(7):650-7). This article describes and demonstrates the feasibility of collecting and assaying nociceptive and inflammatory mediators in surgical wounds at specific time points. The lack of significant correlations between serum and wound levels shows the importance of determining site-specific release if surgical wounds and localized pathologies are to be studied.

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds

A technique to collect and measure surgical wound biochemical mediators at specific time points.

Coleta e medição Mediadores nociceptiva e inflamatória em feridas cirúrgicas

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. ...

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 ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR-Clip - um método para identificar em toda a Transcriptoma Sítios de Ligação de Proteínas RNA Binding

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.

A general protocol for the use of isothermal titration calorimetry to monitor the binding thermodynamics for biological systems with moderate binding affinities is presented.

Calorimetria isotérmica de titulação para medição de Macromoléculas-Ligand Affinity

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction.  In biological ...

Culturing Primary Rat Inner Medullary Collecting Duct Cells

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion.
Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories.

Arginine-vasopressin (AVP) controls fine-tuning of body water homeostasis through facilitating water reabsorption by renal principal cells. Here, we present a protocol for the cultivation of primary rat inner medullary collecting duct cells suitable for the elucidation of molecular mechanisms underlying AVP-mediated water reabsorption.

Cultura primária Rat coletor medular interno células dos ductos

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP ...

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

Titulação Bioquímica de glicogênio<em&gt; In vitro</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is achieved by reverse transcription of the RNA of interest using specific fluorescently labeled primers and subsequent analysis of the resulting cDNA fragments by denaturing polyacrylamide gel electrophoresis. Simultaneously, a traditional Sanger sequencing reaction is run on the gel to map the ends of the cDNA fragments to their exact corresponding bases. In contrast to 5&#39;-RACE (Rapid Amplification of cDNA Ends), where the product must be cloned and multiple candidates sequenced, the bulk of cDNA fragments generated by primer extension can be simultaneously detected in one gel run. In addition, the whole procedure (from reverse transcription to final analysis of the results) can be completed in one working day. By using fluorescently labeled primers, the use of hazardous radioactive isotope labeled reagents can be avoided and processing times are reduced as products can be detected during the electrophoresis procedure.
In the following protocol, we describe an in vivo fluorescent primer extension method to reliably and rapidly detect the 5&#39; ends of RNAs to deduce transcriptional starting points and RNA processing sites (e.g., by toxin-antitoxin system components) in S. aureus, E. coli and other bacteria.

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.

Fluorescência Baseado Técnica Extensão Primer para determinar Transcriptional pontos de partida e de clivagem Sites de RNases<em&gt; In Vivo</em

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is ...

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 ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.