Este trabalho foi financiado por doações do Instituto Nacional de Saúde RO1-GM085130 e National Science Foundation MCB0929394. Agradecemos ao Dr. Marion Schmidt por sua hospitalidade e por nos permitir filme em seu laboratório.

1. Preparação dos Reagentes Footprinting <ol><li> Preparar um tampão de reação 10x, contendo 100 mM cacodilato de sódio, 1 mM EDTA, e 1 M KCl. Ajustar o pH para 7,4. Filtrar o tampão 0,2 mM usando um dispositivo de filtro de acetato (Nalgene). Observação: não pipeta RNA diretamente em tampão 10x. </li><li> Prepare a mistura de reacção de titulação para cada reação, como indicado na Tabela 1. O volume da mistura de titulação (1x buffer e Mg (II) na concentração desejada) deve ser de 90 mL, antes de adicionar 10μl de RNA em tampão 1x. </li><li> Preparar um tampão de digestão RNAse T1 contendo Uréia 6.63M, citrato de sódio 20mM, EDTA 1mM, 0,25 mcg / mL tRNA, cyanol xileno 0,025% e 0,025% azul de bromofenol. Esse buffer pode ser armazenado a 4 ° C por até 6 meses. </li><li> A reação Fenton requer preparada soluções aquosas de 250 mM Fe (NH 4) 2 (SO 4) 2, 250 mM EDTA e 500 mM ascorbato de sódio. As concentrações mencionado no capítulo 10,5 são ideais para experimentos de equilíbrio em tampão cacodilato de sódio, como que não limpam radicais e diversas concentrações de íons divalentes. </li><li> Reagentes para a reação footprinting peroxidativo (1.5.1) e oxidativo (1.5.2). <ol><li> A reação Fenton peroxidativo requer preparada soluções aquosas de 100 mM solução Fe-EDTA, e 50 mM ascorbato de sódio e 1,5% H 2 O 2. A solução Fe-EDTA é preparada pela mistura Fe (NH 4) 2 (SO 4) 2 com EDTA a ter 1,1 vezes mais excesso de EDTA de ferro. </li><li> Pouco antes de iniciar a reação de clivagem oxidativa (3.4.2), criar a mistura de reacção de Fenton, combinando 2 l da Fe (NH 4) 2 (SO 4) 2 (250 mM), 2,2 mL de EDTA (250 mM), 62,5 mL ascorbato de sódio (500 mM), 22,5 mL tampão de reação 10x e 135,5 mL de H 2 O para alcançar a concentração final na mistura de reação footprinting (3.4.2) de 0.10 mM, 0,11 mM e 6,61 mM, respectivamente. </li></ol></li></ol> 2. Preparação para o RNA Experiment Footprinting <ol><li> RNA podem ser produzidos pela norma in vitro a transcrição dos modelos de DNA 6 ou, se menor de 50 nucleotídeos, ser adquirido (por exemplo, Integrated DNA Technologies, www.idtdna.com). Siga os procedimentos de limpeza recomendado laboratório para manter a integridade da amostra de RNA durante as experiências footprinting. 7 </li><li> Desfosforilação 8 de RNA transcrito in vitro é necessário antes de 32 P rotulagem do terminal 5 &#39;do RNA. 10 pmol de RNA são finais marcados por kinasing. 9 A solução deve ser nublado após a inativação da fosfatase sucesso. </li><li> Purificar RNA marcado radioativamente usando eletroforese em gel desnaturante. O RNA é recuperada por dois subseqüentes gel extrações utilizando acetato de sódio 0,3 M (pH 5,5). RNA é precipitado através da mistura de 0,5 ml de solução aquosa com 1 ml de etanol e posteriorincubação por 1 minuto em gelo seco. Rotação da amostra a 4 ° C, 12500 rpm para 0,5 hr. Desprezar o sobrenadante e lavar RNA pellet com etanol 70%. Remover o sobrenadante após a centrifugação adicional e seco pellet no vácuo. </li><li> Dissolver a 32 amostras P-rotulados RNA em 330 ul de 1x 30 mL Buffer.Pipet da solução de RNA em um tubo de reação, precipitado com etanol e seco RNA no vácuo; manter este tubo para gerar a escada de referência (passo 3.5). Desnaturar a solução tamponada de RNA por aquecimento a 95 ° C por 2 min. Arrefecer as amostras à temperatura ambiente por 15 min e dar um giro rápido para derrubar o condensados ​​nas tampas dos tubos na solução. </li></ol> 3. O Experimento Footprinting <ol><li> O pente eletroforese determina o número máximo de pontos de dados por gel analítica. Usamos a 30 comb bem. A escada de referência ocupa 2 poços e um controle uncleaved. Prepare 27 tubos de reação. O Mg final (II) concentrations devem ser uniformemente espaçados em uma escala logarítmica que mede ordens de magnitude em torno do ponto médio de titulação. </li><li> Separadamente, incube a RNA eo Mg (II) de soluções a 50 ° C por 5 min. Misture 10 ml de solução de RNA com 90 mL da correspondente quantidade de Mg (II) em 1x buffer para chegar a um volume final de 100 l. Incubar as soluções por 30 min a 50 ° C. </li><li> Deixe as soluções equilibrar a 25 ° C por 1 hora ao dobrar ocorre. </li><li> Peroxidativo (3.4.1) ou oxidativo (3.4.2) reação pegada radical hidroxila. <ol><li> Iniciar a reação peroxidativo colocando gotas de 2 l Fe-EDTA (100 mM), 2 mL de sódio L-ascorbato (50 mM) e 2 mL H 2 O 2 (1,5%) separados uns dos outros na parte superior interna da reação tubo contendo a solução de RNA e iniciar a reação footprinting pela mistura vigorosa. Parar a reacção após 15 segundos, adicionando 300 mL de etanol frio absoluto. Ligue os tubos de 3-5 vezes. Lavar precipitado, esecar o pellet como no passo 2.4. </li><li> Iniciar o footprintingreaction hidroxila oxidativo radical pela adição de 5 mL da mistura preparada reação de Fenton (1,6). Incubar por 30 min a 25 ° C. Adicione 300 ml de etanol absoluto frio para saciar a reação e misture girando os tubos de 3-5 vezes. Precipitado, lavar e secar o pellet, conforme indicado no passo 2.4. </li></ol></li><li> Gerar uma RNase T1 amostra de referência digerir acordo com os procedimentos padrão de 10 usando a solução preparada na etapa 1.3. </li><li> Dissolver pellets de RNA em 8 mL de gel loading dye II (Ambion) e RNA é ressuspendido confirmar usando o contador Geiger. </li><li> Prepare uma desnaturação, gel de poliacrilamida 8% seqüenciamento de acordo com protocolos padrão. 11 amostras de carga, incluindo duas referências e um controle. Separar fragmentos de RNA de 60 W - 75 W por 2,5 hr. Expor gel seco a uma tela de fósforo de armazenamento durante a noite. Varredura tela de fósforo com um sistema de imagem para autoradiografia filmless, por exemplo,. Tempestade 865 (GE Healthcare) ou Typhoon (GE Healthcare). Transferência de arquivos de imagem gel para o seu computador. </li></ol> 4. Análise de Dados <ol><li> A reatividade radical hidroxila de cada nucleotídeo é determinada pela quantificação da intensidade de cada banda no gel analítica. Baixar, instalar e abrir SAFA, um fácil de usar software de código aberto para instalação de banda única e quantificação. 12, 13 Siga as instruções do manual do usuário. Resumidamente, a seqüência de RNA de carga como. Txt seguido da foto como gel. Gel arquivo. Ajustar intensidades banda, define as pistas, escolha uma pista de âncora, e realizar um alinhamento gel. Atribuição de faixas de nucleotídeos ocorre em referência a RNase escada digestão T1. Quantificar as integrais banda e usar o recurso de normalização / colorplot para normalizar e atribuir resíduos potencialmente invariantes. Salvar a saída como um arquivo txt.. </li><li> SAFA gera uma planilha contendo colunas representando pistas sobre o gel e linhas representando os emgrados densidade de banda das bandas individuais correspondentes ao fragmento de RNA. Primeiro, identificar as zonas de protecção que apresentam uma notável mudança na acessibilidade do solvente através da comparação do perfil de proteção derivado da amostra não Mg (II) com o perfil do ponto final de concentração Mg (II) (50 mM em nosso exemplo). Quanto menor o valor, mais protegido do nucleotídeo é contra o ataque dos radicais hidroxila e vice-versa. </li><li> Criar isotermas dobrar da banda normalizado integrais de indivíduo ou grupos de nucleotídeos em relação Mg concentração (II). As isotermas são individualmente escalado para saturação fracional por fi = L + (U - L) • <img src="files/ftp_upload/3244/3244y.jpg" alt="Y" /> onde f denota a densidade integrada da banda (s) que está sendo analisado, L e U representam os limites inferior e superior para a transição e <img src="/files/ftp_upload/3244/3244y.jpg" alt="Y" /> é a saturação fracionada. 14 Os dados estão aptos usando um l não-linearleste programa de análise de quadrados (Usamos tanto Origem (Origin) ou GraphPad (GraphPad Software, Inc.).) com a equação de Hill (1), <img alt="Equação 1" src="/files/ftp_upload/3244/3244eq1.jpg" /> onde K d é o equilíbrio constante de dissociação, [M] corresponde à concentração da variável que media a reação de dobramento, Mg (II), neste exemplo, e n H é o coeficiente de Hill. Este procedimento escalas a transição para a saturação fracionada, determina o seu ponto médio e fornece um teste fenomenológica de saber se é uma transição sigmoidal. Isotermas derivadas de seqüenciamento géis (Figura 3A), que foram analisadas por SAFA (Figura 3B) e ajuste como descrito acima são mostrados na Figura 3C. As isotermas de saturação fracional mostrado na Figura 3 foram gerados pela escala em uma planilha os valores gerados pela SAFA contra os melhores se encaixam limites superior e inferior (U e L), utilizando a fração equação = valor / (U - L) - L / (U - L). </li></ol> 5. Resultados representativos: A Figura 3 mostra resultados representativos da P4-P6 RNA • experimentos footprinting OH. A imagem gel (à esquerda) indica que uma clivagem de fundo) é mínimo (faixa direita), b) atribuição de único nucleotídeo é possível devido a bandas bem definidas na pista T1 (cliva RNase T1 em cada G), e c) o radical hidroxila induzida fragmentação do RNA é bem acima do fundo. A transição de baixa para alta Mg (II) é afiliado com a diminuição da densidade de banda integrado de individuais e grupos de faixas indicando a formação de RNA estrutura terciária. Individuais ou grupos de nucleotídeos contíguos cuja clivagem alterações concomitantemente são referidos como uma &quot;proteção&quot;. As proteções • OH característica do Mg (II) mediada dobramento da RNA P4-P6 perto correspondem às regiões inacessíveis solvente da molécula observada em sua estrutura cristalina. 15 Dobrado moléculas RNA c<html> um tem extensões diferentes (isto é, o quão perto a fundo a banda declínio densidades) de proteção de contato superior que não têm sido correlacionados com fenômeno estrutural ou dinâmico clara. Assim, algumas moléculas de RNA vai mostrar bem definidas transições estruturais, como mostrado na Figura 3, e alguns não. Intensidades banda são quantificados e normalizados pela SAFA análise 12 (Figura 3B). A saída é uma trama &#39;térmico&#39; visualizando o relativo grau de protecção contra o • OH. A transição de cores descreve a mudança de acessibilidade em cima disso Mg (II). Branco para vermelho ou azul mostra nucleotídeos mais acessível ou mais protegidos, respectivamente. Cada grau de sombreamento é afiliado com um valor numérico que pode ser plotado como uma curva de proteção (Figura 3C) e analisados ​​por um modelo de ligação, tais como a equação de Hill (1). O equilíbrio constante de dissociação afiliada à Proteção 153-155 é aproximadamente o dobro do valor correspondente de Proteção 163-164. ONTEÚDO &quot;&gt; <img alt="Figura 1" src="/files/ftp_upload/3244/3244fig1.jpg" /> Figura 1. Produção de radicais hidroxila e P4-P6 dobrar RNA. A) Fe (II) catalisa a geração de radicais hidroxila a partir de peróxido de hidrogênio. Ascorbato reduz Fe volta (III) a ferro. B) Na ausência de Mg (II), nenhuma estrutura P4-P6 terciário é formada, permitindo que os radicais hidroxila para penetrar e se unir todas as posições backbone acessível. Além do Mg (II) inicia dobrar de P4-P6, permitindo apenas a espinha dorsal do solvente acessíveis a ser clivada pelos radicais hidroxila. <img alt="Figura 2" src="/files/ftp_upload/3244/3244fig2.jpg" /> Figura 2. Esboço para uma experiência pegada radical hidroxila. A Dephophorylation) e 32 P rotulagem 5&#39;-final da RNA. B) Purificação de 32 P-rotulados RNA em gel de poliacrilamida desnaturante. C) Excisão da banda de RNA, extração de RNA subseqüentes, e precipitação de etanol. D Prefolding) edobramento de RNA. E) A adição de recém-preparada mistura de reação Fenton para gerar os radicais hidroxila. F) separação fragmento de RNA por eletroforese desnaturante em gel de poliacrilamida. G) A quantificação de fragmentos de RNA por software SAFA. <img alt="Figura 3" src="/files/ftp_upload/3244/3244fig3.jpg" /> Figura 3. Análise de fragmentos de RNA após a reação peroxidativo footprinting Fenton. (A) O RNA foi exposto a radicais hidroxila e os produtos de dissociação foram resolvidas usando eletroforese em gel de poliacrilamida desnaturante (PAGE). A imagem mostra produtos de dissociação peroxidativo com o aumento da concentração de Mg (II). Pistas de referência e controle são rotulados. Este arquivo de imagem é a entrada para o programa SAFA, que quantitates o volume de cada banda. (B) O enredo &quot;térmico&quot; gerado pela SAFA. (C) A planilha de valores relativos a densidade de banda integrado para a saída número de nucleotídeos de SAFA é analisada por um modelo de vinculação, como o equati Colinaem (1). Regiões de proteção foram escolhidos de acordo com o aumento da densidade de banda integral como uma função da concentração de Mg (II). A d K é a concentração de Mg (II) em que metade do RNA é dobrada no local está sendo monitorado. O coeficiente de Hill, n H, é uma medida da inclinação da curva que dá informações sobre a cooperatividade de ligação e oferece um menor estimativa para o número de íons de magnésio envolvidos na dobragem deste site particular. <img alt="Tabela 1" src="/files/ftp_upload/3244/3244table1.jpg" /> Tabela 1. Volumes Representante para a geração de amostras de RNA contendo diferentes Mg (II) as concentrações.

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Footprinting radical hidroxila é uma ferramenta valiosa para avaliar a área de superfície acessível solvente de ácidos nucléicos. Formação qualitativa e quantitativa da estrutura terciária 14 pode ser seguido em função de parâmetros como tipo e concentração de íons, pH, temperatura, proteínas de ligação ou dobrar co-fatores. A combinação irresistível de um protocolo para a frente e de baixo custo e acessibilidade resultante de solvente e informações dobrar em um nível de nucleotídeo ?...

<table border="1">
	<tbody>
		<tr>
			<th>Name</th>
			<th>Company</th>
			<th>Cat#</th>
		</tr>
		<tr>
			<td>Sodium Cacodylate
			(Caution! Toxic)	 	
			</td>
			<td>Sigma</td>
			<td>C4945-25g</td>
		</tr>
		<tr>
			<td>EDTA (0.5 M)</td>
			<td>Ambion</td>
			<td>AM9260G</td>
		</tr>
		<tr>
			<td>DEPC treated water</td>
			<td>Ambion</td>
			<td>AM9915G</td>
		</tr>
		<tr>
			<td>Sodium Acetate (3 M)</td>
			<td>Ambion</td>
			<td>AM9740</td>
		</tr>
		<tr>
			<td>MgCl2 (1 M)</td>
			<td>Ambion</td>
			<td>AM9530G</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Ambion</td>
			<td>AM9902</td>
		</tr>
		<tr>
			<td>Sodium Citrate</td>
			<td>Sigma</td>
			<td>W302600</td>
		</tr>
		<tr>
			<td>tRNA</td>
			<td>Sigma</td>
			<td>R-7876</td>
		</tr>
		<tr>
			<td>Sodium-L-ascorbate</td>
			<td>Sigma</td>
			<td>A7631-25g</td>
		</tr>
		<tr>
			<td>Fe(NH4)2(SO4)2 . 6 H2O</td>
			<td>Sigma</td>
			<td>F1543-500g</td>
		</tr>
		<tr>
			<td>RNase T1</td>
			<td>Fermentas</td>
			<td>EN0541</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide (30%)</td>
			<td>Sigma</td>
			<td>349887</td>
		</tr>
	</tbody>
</table>

monitoring equilibrium changes in rna structure by 'peroxidative' and 'oxidative' hydroxyl radical footprinting

Moléculas de RNA desempenham um papel essencial na biologia. Além de transmitir a informação genética, RNA pode dobrar em exclusivo estruturas terciárias cumprindo um papel biológico específico como ligante regulador, ou catalisador. Informações sobre a formação de contato superior é essencial para compreender a função das moléculas de RNA. Radicais hidroxila (OH •) são sondas única da estrutura dos ácidos nucléicos, devido à sua alta reatividade e tamanho pequeno. 1 Quando utilizado como uma sonda pegada, os radicais hidroxila mapa da superfície acessível solvente do backbone fosfodiéster do DNA e RNA 1 2 com tão fina como a resolução de nucleotídeo único. Footprinting radical hidroxila pode ser usado para identificar os nucleotídeos dentro de uma superfície de contato intermolecular, por exemplo, DNA-proteína-1 e RNA-proteína complexos. Equilibrium 3 e 4 transições cinética pode ser determinada através da realização de pegada radical hidroxila em função de um solutina variável ou tempo, respectivamente. Uma característica fundamental da pegada é que a exposição limitada à sonda (por exemplo, &#39;single-hit cinética &quot;) resulta na amostragem uniforme de cada nucleotídeo do polímero. 5 Neste artigo de vídeo, usamos o domínio P4-P6 da ribozima Tetrahymena para ilustrar RNA preparação de amostras e determinação de um (II) mediada por Mg isotermas de dobradura. Descrevemos a utilização do protocolo pegada bem conhecido radical hidroxila que requer H 2 O 2 (nós chamamos este protocolo &quot;peroxidativo &#39;) e um valor, mas não muito conhecido alternativa, que usa naturalmente O 2 dissolvido (chamamos isso de&quot; o protocolo oxidativo &quot;). Uma visão geral da redução de dados, transformação e procedimentos de análise é apresentado.

Watch this Scientific Journal Video about Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting at JoVE.com

Monitoring Equilibrium Changes in RNA Structure by &#039;Peroxidative&#039; and &#039;Oxidative&#039; Hydroxyl Radical Footprinting

Este protocolo descreve como quantificar o Mg (II) dependente da formação de RNA estrutura terciária através de dois métodos de pegada radical hidroxila.

Monitoramento de mudanças na estrutura Equilibrium RNA por &quot;peroxidativo&quot; e &quot;oxidativo&quot; Footprinting Radical Hidroxila

RNA molecules play an essential role in biology. In addition to transmitting genetic information, RNA can fold into unique tertiary structures fulfilling ...

monitoring-equilibrium-changes-rna-structure-peroxidative-oxidative

Research

JoVE Journal

Biology

Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting

14.2K visualizações.  Hunter College. Este protocolo descreve como quantificar o Mg (II) dependente da formação de RNA estrutura terciária através de dois métodos de pegada radical hidroxila.

Vídeo: Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

Métodos única molécula para acompanhar as alterações nas propriedades elásticas bicamada

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

Biologia

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Mudanças Dinâmicas de monitoramento dos níveis de cálcio mitocondrial durante a apoptose através de um sensor de cálcio geneticamente codificado

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

Transcrição in vitro e nivelamento de Gaussia mRNA luciferase Seguido por HeLa transfecção celular

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

A longo prazo de Teste de toxicidade letal com o crustáceo Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Isolamento de cromatina por purificação de RNA (CHIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Detecção de RNA viral por fluorescência<em&gt; In situ</em&gt; Hybridization (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

RNA Secundária Previsão Estrutura Usando SHAPE de alta capacidade

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

Reconstituição funcional e atividade do canal Medidas de purificada tipo selvagem e mutante CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.

Receptor trafficking modulates signaling and cell responsiveness to ligands and is, itself, responsive to cell conditions, including ligand-induced signaling. Here, we describe a powerful and flexible technique for quantitatively assessing drug-induced receptor trafficking using immunolabeling and colocalizational analysis.

Rastreando alterações induzidas pela droga em Receptor de Pós-internalização Tráfico de Análise Colocalizational

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fecal Análise de glicocorticóides: Non-invasive Monitoring Adrenal em eqüídeos

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...