This work was supported by NIH grant GM085232 and an Irma T. Hirschl/ Monique Weill-Caulier Scholar Award (to A.A.).

1. Preparação Lipid <ol><li> Alíquota os lípidos quantidade desejada para um tubo de vidro transparente. Use E. coli extracto de lípidos polares, mas a maioria das composições de lipidos podem ser usados. Se os lípidos são na forma de pó, ressuspender em clorofórmio a uma concentração de 20 mg / ml. Secar os lípidos à temperatura ambiente sob atmosfera de N 2 gasoso até todo o solvente se ter evaporado. </li><li> Ressuspender os lipídios em pentano e secá-lo novamente um fluxo constante de gás N 2 para remoção de vestígios que sobraram de clorofórmio. Seco até todo o solvente tenha evaporado. Enquanto a secagem, rodar suavemente o tubo de modo a que o lípido é distribuído numa película uniforme que cobre o terço inferior do tubo em vez de uma formação de uma massa densa na parte inferior. </li><li> Adicionar o tampão de reconstituição contendo detergente (KCl 300 mM, ácido cítrico 50 mM, 25 mM de K 2 HPO 4, pH 4,5, CHAPS a 35 mM) para dissolver os lípidos. Usar outros detergentes suaves para este passo, se a proteína é mal tolerante para CHAPS. </li><li> Resuspend os lipídios a clareza usando um banho de água sonicator. Mergulhar o tubo no centro do banho de ultra-sons de água à potência máxima de curta duração (30 sec-1 min) com pulsos de curta duração (30 sec-1 min) faz uma pausa. A solução deve ficar clara. NOTA: O grau final de clareza obtida depende da composição do lípido específico utilizado. Alguma variação lote-a-lote neste passo, mesmo entre lípidos nominalmente idênticas, também é possível. Turvação residual é devido à dispersão da luz pelas partículas grandes em suspensão, que podem ser removidos por centrifugação. </li><li> Incubar os lípidos ressuspensas a TA durante 20-60 min. </li></ol> 2. proteolipossoma Formação NOTA: Várias estratégias podem ser empregues para inserir o detergente-proteína solubilizada em lipossomas. Para CLC-EC1 diálise funciona bem e é, por conseguinte, o método de escolha 6,9,10. <ol><li> Adicionar a proteína purificada para a desejada proteína e lípido (P / L) razão (expressa em ug o<html> f proteína / mg de lípido). A escolha da relação P / L depende do objectivo da experiência. <ol><li> Se o objectivo é o de avaliar a actividade da proteína de interesse, em reconstituir alta P / L para maximizar o sinal, uma vez que cada liposoma contém múltiplas cópias da proteína. Para quantificar a actividade e / ou a taxa de transporte unitário da proteína, reconstitua num regime de diluição de Poisson em baixo P / L, de modo que cada vesícula contém, em média, uma proteína. Veja o Passo 7 e Discussão para uma análise mais profunda de quando usar cada regime. </li></ol></li><li> Para determinar os valores de P / L óptimas para os vários esquemas, realizar uma titulação completa da actividade como uma função da concentração de proteína 8,11-13. No entanto, para qualquer proteína para a qual o peso molecular (PM, expressa em kDa) da unidade activa é conhecido os seguintes valores de P / L podem ser utilizados como guesstimates iniciais: pg &quot;/&gt; <img alt="Equação 2" fo:content-width="1.5in" src="/files/ftp_upload/52369/52369eq2.jpg" /></li><li> Coloque a mistura de proteína e lípido num tubo de diálise pré-humedecido ou uma cassete. Colocar o dispositivo de diálise num copo contendo 500-1,000x o volume do tampão de reconstituição lípido (isto é, um tampão de L para 1 ml de ressuspensão lipídica) sob agitação suave. <ol><li> Mudança de tampão de 3-4 vezes em intervalos de&gt; 3 h. Em cada mudança solução, lava-se a cassete / tubo e o copo com água destilada e enche o recipiente com tampão fresco a reconstituição. Incluir 1-2 S / N intervalos para a mudança solução. O número exacto e duração da solução muda depende do lípido exacta e detergentes utilizados. </li></ol></li><li> Após a última etapa é concluída, remova os lipossomas a partir do navio de diálise, aliquotar-los em tubos e flash congelá-los em N 2 líquido ou congelar a -80 ° C. </li></ol> 3. A gravação Set-up &lt;p class = &quot;jove_content&quot;&gt; NOTA: A gravação de set-up (Figura 2A) consiste de duas câmaras (cilindros de fundo plano, ~ 3-4 ml de volume), um Cl - (veja abaixo) eletrodo, um medidor de pH com um análogo ou saída digital elétrica, um digitalizador e um computador com um software de aquisição apropriado. <ol><li> Ligue o Cl - eléctrodo para o medidor de pH, a saída do medidor de pH para o digitalizador que está ligado ao computador. Inserir o eléctrodo de referência em uma câmara e o fio recodificação no outro (Figura 2B). NOTA: A polaridade dos fios é correto se AV Cal (ver Passo 6.4 e 7.2) é positivo. </li><li> Coloque as câmaras numa placa de agitação com uma barra de agitação na câmara de gravação (Figura 1B). Certifique-se a barra de agitação não toca o eletrodo. </li><li> Ligar as câmaras que utilizam uma ponte de agar (Figura 2B) (KCl 100 mM e 2% de agarose). Guarde as pontes Agar em KCl 100 mM. </li&gt; </ol> 4. Preparação do Cl - Eléctrodo <ol><li> Tome um eletrodo de pH não-funcionamento de idade e -possibly-. Quebrar o revestimento de vidro para revelar completamente os fios de prata. </li><li> Retire cuidadosamente o revestimento dos fios de prata, usando uma lâmina de bisturi. Limpe os fios usando grandes quantidades de H 2 O e EtOH. </li><li> Lugar numa solução saturada de FeCl 3 (ou cloro) até fios são cobertos com um revestimento escuro uniforme de AgCl. </li></ol> 5. Preparação de Unilamelares Vesicles <ol><li> A noite antes das experiências, inchar 1 g de Sephadex G-50 em grânulos de 15 ml de tampão externo (1 mM KCl, 150 mM de K 2 SO 4, ácido cítrico 25 mM, pH 4,5) com agitação suave (não agite) para pelo menos 3 horas à temperatura ambiente antes da sua utilização. Cada experiência requer ~ 3 ml de pérolas inchadas. Manter os grânulos inchados a 4 ° C durante alguns dias no máximo. </li><li> Embora os lipossomas são descongelar à temperatura ambiente, verter em cada coluna de ~ 3 ml de inchados G-50 grânulos. Deixe-os secar por fluxo de gravidade na RT; isso geralmente leva 1-2 min. </li><li> Preparar vesículas unilamelares por extrusão dos proteolipossomas 11 vezes através de um mini-extrusora utilizando um ponto de corte de 0,4 m de Teflon. </li><li> Inserir a coluna em um tubo de plástico de fundo redondo. Gire as colunas para remover o excesso de solução. Centrifugar durante 20-30 segundos a 1400 x g numa centrífuga clínica. </li><li> Descartar escoamento, lugar coluna num tubo de vidro de 13 x 100 mm e adicionar 100 ul de vesículas extrudidas para a coluna. Rotação coluna durante 1 min a 500 xg numa centrifugadora clínica. </li><li> Recolhe ~ 200 ul de fluxo de passagem que vão ser adicionados à câmara de registo no passo 6.5. </li></ol> 6. Efluxo Medição <ol><li> Coloque 2 ml de KCl 100 mM na câmara (terra) de referência, e 1,8 ml de tampão externo (1 mM KCl, 150 mM de K 2 SO 4, 25 mM de ácido cítrico, pH 4,5) para a câmara de registo. O ligeiro desequilíbrio osmótico entre o internoe soluções externas não afeta </li><li> Inicie o programa de aquisição. Deixe a linha de base equilibrar, isso pode levar alguns minutos. </li><li> Uma vez que o sinal atinge uma linha de base estável (os lipossomas são preparados e as colunas de secar), iniciar a gravação (Figura 3A). </li><li> Adicionar 15 ul de uma solução 10 mM de KCl para calibrar o sistema (Figura 2A). </li><li> Adicionar lipossomas. Um pequeno salto pode ser visível devido à remoção incompleta de Cl externo - a partir das proteoliposomes (Figura 3A). Espere até a linha de base se estabilize. </li><li> Adicionar valinomicina (1 ml a 1 mg / ml em EtOH) para iniciar o efluxo (Figura 3A). </li><li> Deixe o efluxo de correr o seu curso de tempo, até que se planaltos (Figura 3A). </li><li> Adicionar 40 ul de 1,5 M β-octilglucósido (β-OG), preparado no tampão externo para dissolver todos os liposomas (Figura 3A). Fim da gravação. </li></ol> 7. Data Análise <ol><li> Exportar o arquivo de rastreamento a partir de um formato ASCII ou texto e importá-lo para o programa de análise de escolha. </li><li> Meça a tensão nos seguintes pontos (Figura 3A): No início da gravação (V 0); Após a adição do impulso de calibração (V cal); Após a adição dos lipossomas (V lipo); No final do efluxo (V fin); Após a adição de detergente (V tot); Linha de base, de modo que as tensões V 0 = 0. </li><li> Utilizar a equação de Nernst-prancha para determinar o valor experimental de <img alt="Equação 3" fo:content-width=".3in" src="/files/ftp_upload/52369/52369eq3.jpg" /> medindo <img alt="Equação 4" fo:content-width="3in" src="/files/ftp_upload/52369/52369eq4.jpg" /> Onde Vol Cal é o volume do impulso de calibração em ul, 1,800 é o volume da câmara no início da experiência, expresso em1; l, [Cl] cal / in são o Cl - concentrações do pulso de calibração e do buffer externo em mM e AV cal é o salto em mV medido após o pulso de calibração. NOTA: Esta determinação empírica de α serve a três propósitos: ele garante que o sistema está respondendo corretamente, oferece uma medida da consistência da resposta do instrumento entre os experimentos, bem como para verificar se não há erros foram cometidos na tomada de solução. </li><li> Calcular a concentração de Cl após cada passo da seguinte forma: <img alt="Equação 5" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq5.jpg" /> <img alt="Equação 6" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq6.jpg" /> <img alt="Equação 7" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq7.jpg" /> O factor de 3/400 vem da diluição do impulso de calibração 15 ul no volume final de 2.000 mL. </li><li> Calculcomeu as mudanças na [Cl] em cada etapa, ΔCl lipo / fin / tot. </li><li> Converter V (t) em Cl (t) com <img alt="Equação 8" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq8.jpg" /> Diretamente determinar os valores [CL] nos pontos críticos e compará-los com os valores calculados como um controlo interno. </li><li> Normalizar o traçado de modo que a lipo Cl rel = 0 e Cl tot rel = 1 <img alt="Equação 9" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq9.jpg" /></li><li> Montar o curso de tempo de efluxo com a seguinte equação: <img alt="Equação 10" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq10.jpg" /> Onde L é a taxa de Cl - vazamento que é determinada experimentalmente medindo Cl - efluxo de lipossomas livres de proteínas preparadas nas mesmas condições e τ é a constante de tempo do processo. </li><li> Calcular v, a velocidade inicial: Onde ΔCl barbatana é expressa em milimolar e V é o volume da câmara em ul. </li><li> Calcular a taxa de transporte / condutância <img alt="Equação 12" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq12.jpg" /> Em que p é a relação P / L, MW é o peso molecular do complexo activo, expresso em kDa </li></ol>

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The authors declare no competing financial interests.

Nós descrevemos um protocolo detalhado para medir Cl - transporte mediado pelos canais de ânion seletivo purificadas ou transportadores reconstituídos em lipossomas. O exemplo utilizado foi o procariotas H + / Cl - Trocador CLC-EC1. No entanto, a metodologia pode ser facilmente adaptada para estudar canais fechados por ligandos 12,13,15, 11,12 tensão, ou que ostentam diferente selectividade aniónico 15,16 substituindo o Ag: AgCl com um adequado para o ião sob ...

Nas últimas duas décadas, a capacidade para sobre-expressar e purificar proteínas transportadoras de membrana aumentou dramaticamente: canais de iões, os transportadores principal e secundário são rotineiramente purificado a partir de sistemas de expressão heterólogos, bem como as fontes naturais. Novas abordagens para monitorar expressão, melhorar e facilitar a extração e melhoram a estabilidade dessas proteínas estão sendo constantemente desenvolvidos 1-5. Estes avanços tecnológicos têm sido fundamentais para desencadear a explosão de informações estruturais em nível atômico em proteínas de membrana, que, por sua vez, reforçada a nossa compreensão das bases estruturais da sua função. Em contraste, a nossa capacidade para sondar as propriedades funcionais das proteínas purificadas não aumentaram com a mesma velocidade, de modo que, em alguns casos, a informação estrutural de alta resolução é acompanhada pelos dados funcionais qualitativos, limitando assim a capacidade de testar quantitativamente previsões baseadas em estrutura. Assim, o desenvot de ensaios funcionais quantitativos e generalizáveis ​​é um passo fundamental para a elucidação das bases mecanicistas da função da proteína de membrana. Descrevemos aqui um ensaio de efluxo que pode ser utilizado para determinar quantitativamente as propriedades funcionais da purificados e reconstituídos Cl - canais e transportadores. Os princípios subjacentes a ensaio pode ser generalizada para uma variedade de sistemas de transporte, bem como para o transporte de proteínas não-iônicos. Os lipossomas são reconstituídos com purificados Cl - / canal transportadores, na presença de um grande gradiente de Cl - (Figura 1A, B). Cl - efluxo é iniciada pela adição de um ionóforo para permitir fluxo de contra-ião, no nosso caso, o K + ionóforo valinomicina, que desviam a tensão estabelecida pela Cl - gradiente e definir o potencial de membrana inicial do potencial de equilíbrio de K + 6,7. Sem tele não ionóforo Cl líquido significativo - efluxo ocorre, uma vez que é impedida pela geração de um potencial transmembranar. Os dados são descritos quantitativamente por dois parâmetros mensuráveis ​​(Figura 1C): τ, a constante de tempo de efluxo de Cl -, e f 0, a fracção de lipossomas que não contêm uma proteína activa. De τ 0 e f o Cl unitário - a taxa de transporte, a fracção de proteínas activas e a massa molecular do complexo activo pode ser derivado 8. A técnica é ilustrada aqui, usando proteoliposomes reconstituídos com CLC-EC1, um bem caracterizado CLC-tipo H + / Cl - trocador de estrutura e função conhecida. Este ensaio é prontamente generalizado para canais ou transportadores com diferentes selectividade iónica ou cuja actividade depende da presença de tensão e / ou ligantes. Além disso, este ensaio pode ser usado para determinar se as moléculas pequenas afectar directamente a proteína reconstituída,para quantificar os efeitos destes compostos e composição como membrana ou reagentes modificadores de lípidos afectar a função dos canais reconstituídos e transportadores.

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			<td height="21" style="height:21px;width:372px;">Name of Reagent/ Equipment</td>
			<td style="width:337px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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			<td height="21" style="height:21px;">Liposomicator, Avanti Polar Lipids Inc.</td>
			<td style="width:337px;">&#160;Avanti Polar Lipids Inc.</td>
			<td>610200</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">IEC Centra CL2 Benchtop</td>
			<td style="width:337px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Orion Research Model 701A Digital pH-mV meter</td>
			<td style="width:337px;"></td>
			<td style="width:119px;"></td>
			<td>These can be found on Ebay.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Non-functional pH probe</td>
			<td style="width:337px;"></td>
			<td style="width:119px;"></td>
			<td>Any pH meter probe with silver wires will work. The glass/plastic coating needs to be removed and the wires cleaned.</td>
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		<tr height="21">
			<td height="21" style="height:21px;">&#160; DI-710 Data Logger</td>
			<td style="width:337px;">DATAQ instruments</td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">WinDAQ acquisition software</td>
			<td style="width:337px;">DATAQ instruments</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Pierce Disposable Plastic Columns, Gravity-flow, 2ml</td>
			<td style="width:337px;">Pierce (Thermo Scientific)</td>
			<td>29922</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">KIMAX Culture Tubes, Disposable, Borosilicate Glass</td>
			<td style="width:337px;">Kimble Chase</td>
			<td>73500-13100</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Extruder Set With Holder/Heating Block</td>
			<td style="width:337px;">Avanti Polar Lipids Inc.</td>
			<td align="right" style="width:119px;">610000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Computer</td>
			<td style="width:337px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
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a proteoliposome-based efflux assay to determine single-molecule properties of cl- channels and transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl- from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl- exchanger, as a model system. The efflux assay can be utilized to study any Cl- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

Nós descrevemos um protocolo detalhado e robusto para medir Cl - transporte mediado por purificada CLC-EC1, um procariota CLC-tipo H + / Cl - trocador, reconstituída em lipossomas. Uma representação esquemática da experiência é mostrado na Figura 3 proteoliposomes reconstituídos com CLC-purificada contendo EC1 e alta - Cl interno. São imersos numa solução de banho que contém baixo Cl -. Sob estas condições Cl líquido - eflu...

Watch this Scientific Journal Video about A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters at JoVE.com

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

A proteolipossoma-Based Efluxo Ensaio para determinar as propriedades de uma única molécula de Cl<sup&gt; -</sup&gt; Canais e Transportadores

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...

a-proteoliposome-based-efflux-assay-to-determine-single-molecule

Research

JoVE Journal

Biology

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

9.7K visualizações.  Weill Cornell Medical College. Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

Vídeo: A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

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

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

Replicação de DNA visualizar Único molécula com microscopia de fluorescência

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

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

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

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

Sistema automatizado para medições única molécula de fluorescência de Superfície-imobilizada Biomoléculas

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

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

A observação direta de Enzimas DNA Replicação Usando um DNA molécula única Alongamento Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

Um ensaio de rastreio fluorescente para identificação Moduladores de Canais GIRK

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

Molécula única imagem da regulação gênica<em&gt; Em vivo</em&gt; Uso de ativação Cotranslational por clivagem (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

Combinando uma única molécula de Manipulação e imagem para o estudo das interações proteína-DNA

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

Estudar DNA Looping por Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

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 ...

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. These transport mechanisms are mediated by the activities of specialized transporter proteins. Specifically,pH dynamics are finely controlled by plasma membrane proton (H+) extrusion systems, such as the Na+/H+ exchanger (NHE) protein family. Despite extensive efforts to study the mechanisms underlying NHE regulation, our current understanding of the biophysical and molecular properties of the NHE family is inadequate because of the limited availability of methods to effectively measure NHE activity. In this manuscript, we used H+-selective electrodes during whole-cell patch clamping recording to measure NHE-induced H+ flux. We proposed this approach to overcome some limitations of typically used methods to measure NHE activity, such as radioactive uptake and fluorescent membrane permeants. Measurement of NHE activity using the described method enables high sensitivity and time resolution and more efficient control of intracellular H+ concentrations. H+-selective electrodes are based on the fact that transporter activity creates an ion gradient in close proximity to the cell membrane. An H+-selective electrode moving up to and away from the cell membrane in a repetitive, oscillatory fashion records a voltage difference that is dependent on H+ flux. While H+-selective electrodes are used to detect H+ flux moving out of the cell, the patch clamp method in the whole-cell configuration is used to control the intracellular ion composition. Moreover, application of the giant patch clamp technique allows modification of the intracellular composition of not only ions but also lipids. The transporter activity of NHE isoform 3 (NHE3) was measured using this technical approach to study the molecular basis of NHE3 regulation by phosphoinositides.

This manuscript describes the applications of proton-selective electrodes and patch clamping methods to measure the activity of proton transport systems. These methods overcome some limitations of techniques commonly used to study proton transport activity, such as moderate sensitivity, time resolution and insufficient intracellular milieu control.

Aplicação de medida de eletrofisiologia para estudar a atividade de transportadores de Electro-neutro

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. ...