Este trabalho foi apoiado pela Fondo Nacional de Desarrollo Científico y Tecnológico (Fondecyt), Chile, projeto 11130263 (para CW), projeto CONICYT, NERC + Programa de Colaboración Internacional PCI-PII20150073 (para CW) e U-inicia a partir do Vicerrectoría de Investigación Universidad de Chile (CW).
Para Tatiana Naranjo-Palma, obrigado pela ajuda na fase inicial deste projecto, Dr. Jorge Babul e Diego Quiroga-Roger para discussão útil, Robert Lesch para revisar o manuscrito e Dirección de ramal de la Facultad de Ciencias Químicas y Farmacêuticas da Universidade do Chile. Graças também ao Marcelo Pérez para edição de vídeo e Neil Stevens para corrigir o texto e Errol Watson para fornecer a voz do narrador.

1. preparação e execução do Gel
<ol><li>Prepare o desnaturação não 12% gel de polyacrylamide gel usando a receita de sódio Dodecil sulfato de sódio-Polyacrylamide Gel eletroforese (SDS-PAGE) encontrados em Sapeka e Russell14 mas usando água em vez de SDS.<ol>
<li>Para preparar a 5 mL de solução de gel, use 1,7 mL de água destilada, 2 mL de acrilamida/bis acrilamida (30/0,8% p/v), 1,3 mL de 1,5 M Tris pH 8,8, 0,05 mL de persulfato de amónio de 10% e 0,002 mL de TEMED.</li>
		<li>Para preparar 2 mL de gel de empilhamento usar 1,5 mL de água, 0,33 mL de acrilamida/bis acrilamida (30/0,8% p/v), 0,25 mL de 1 M Tris pH 6,8, 0,02 mL de persulfato de amónio de 10% e 0,002 mL de TEMED. As dimensões do gel são 7,3 x 8,2 cm x 0,075 cm (l x w x d). Nota: Use um pente bem 15 de 0,75 mm de espessura para a melhor sensibilidade.</li>
	</ol>
</li>
	<li>Adicione as quantidades adequadas de 6x carregando buffer e amostra de DNA para um tubo de microcentrifugadora de 1,5 mL (por exemplo, uso 1 µ l de tampão de carregamento 6 x a 5 µ l de amostra de DNA).</li>
	<li>Coloca o gel na câmara de eletroforese vertical.<ol>
<li>Encha a câmara com 750 mL de 1 tampão de x TAE para executar 2 géis. Para preparar 1 L de 50 x TAE, usar 442 g da base Tris, 57,1 mL de ácido acético glacial e 100 mL de pH de EDTA 0,5 M 8. Adicionar água destilada até um volume final de 1 L.</li>
		<li>Carrega as amostras no gel. Funcione o gel a 100 V por 2 h.</li>
	</ol>
</li>
	<li>Em alternativa, prepare um gel de agarose 1 x TAE usando a receita encontrada na Sapeka14.<ol>
<li>300 ml de 1 x TAE para a câmara de eletroforese horizontal. Funcione o gel por 45 min a 100 V.</li>
	</ol>
</li>
</ol>2. NBT-SG eu coloração (Figura 1).
<ol><li>
Gel do acrilamido coloração.
	<ol>
<li>Preparar as soluções: 0,1% w/v NBT e 1.2 x SG eu. Nota: É possível usar o brometo de etídio, GelRed e SYBR Gold em vez de SG eu, mas eu tive o melhor desempenho de SG.</li>
		<li>Despeje 16,75 mL de 1.2 x SYBR verde eu solução em um recipiente de tamanho apropriado. Nota: Usamos a tampa de uma caixa de ponta da pipeta como um recipiente, e esses volumes são calculados para cobrir o gel usando este recipiente; suas dimensões são 11,6 x 7,6 cm x 2.6 cm (l x w x d).</li>
		<li>Coloque o gel na solução. 3,25 ml de solução de 0,1% w/v NBT dar NBT de 0.2 mM e 1 x (equivalente a 2,0 µM) SG concentrações finais.</li>
		<li>Selar o recipiente com filme plástico e enrole completamente o recipiente com papel de alumínio para bloquear qualquer luz ambiente. Nota: O filme plástico é usado para evitar salpicos e evitar a reação da película de alumínio com a coloração da solução.</li>
		<li>Agitar durante 25 min em um agitador orbital em 60 rpm.</li>
		<li>Remova o alumínio e tampa de plástico. Exponha à luz solar por 90 min máximo. Verificar a cada 5 min até que apareça a banda de interesse.</li>
		<li>Para usar a luz azul, preparar uma protoboard com 3 diodos emissores de luz (LED) e colocar os LEDs 5 mm da superfície do gel (verifique a coloração continuamente, como o tempo necessário dependerá da intensidade da fonte de luz LED).</li>
	</ol>
</li>
	<li>
A coloração do Gel do agarose.
	<ol>
<li>Depois de preparar e executar um agarose 1% gel conforme descrito nas seções 1.4 (as dimensões são 1 x 6,2 cm x 7 cm; l x w x d), siga os mesmos passos como em 2.1 com essas modificações:</li>
		<li>Despeje 41,9 mL de 1.2 x SG eu solução na tampa de uma caixa de ponta da pipeta; suas dimensões são 11,6 x 7,6 cm x 2.6 cm (l x w x d). Adicione 8,1 mL da solução NBT de 0,1%.</li>
		<li>Selar o recipiente com filme plástico e em seguida, enrole completamente o recipiente com papel de alumínio para bloquear qualquer luz ambiente.</li>
		<li>Agitar durante 1 h em um agitador orbital em 60 rpm.</li>
	</ol>
</li>
</ol>3. os dados análise.
<ol><li>
NBT-SG que as curvas de calibração.
	<ol>
<li>Obter imagens de gel de digitalização de 1200 dpi.</li>
		<li>Software aberto para análise de imagem. Endireite as imagens usando um editor de imagem. Execute uma densitometria de imagem.</li>
		<li>Calcule a área sob a curva. Prepare um gráfico do sinal/sinalMax versus concentração de DNA.</li>
	</ol>
</li>
</ol>4. o DNA recuperação.
<ol><li>Preparar um gel de agarose 1% em 1 x TAE usando a receita encontrada na Sapeka14.</li>
	<li>300 ml de 1 x TAE para a câmara de eletroforese horizontal. Carrega 4 µ l de 100 plasmídeo de15 pDJ100 ng / µ l e escada de DNA, seguindo o esquema na Figura 2. Funcione o gel durante 1 h a 100 V.</li>
	<li>Marcar e pesar dois tubos de microcentrifuga de 2 mL.</li>
	<li>Corte o gel no meio para ter duas metades idênticas. Cada um deve conter a escada e o plasmídeo de amostra para comparar as diferentes técnicas de coloração. Uma representação esquemática é na Figura 2.</li>
	<li>Manchar uma metade com NBT-SG eu como na etapa 2.<ol>
<li>Corte a banda do plasmídeo usando um bisturi limpo. Guarde o pedaço de gel em um tubo e pesar.</li>
	</ol>
</li>
	<li>Manchar a outra parte do gel com SG eu com 50 mL de 1 x SG eu solução e agitar lentamente (60 rpm) o gel por 1h na escuridão.<ol>
<li>Coloque o gel em um transiluminador e expor por 2 min. corte a banda do plasmídeo usando um bisturi limpo.</li>
		<li>Guarde o pedaço de gel no outro tubo e pesar. Nota: O protocolo pode ser pausado aqui, mantendo os tubos a-20 ° C.</li>
	</ol>
</li>
	<li>Realize a extração do plasmídeo usando um kit de extração comercial de gel.</li>
</ol>5. plasmídeo normalização.
<ol><li>Em um tubo de microcentrifugadora de 1,5 mL, adicione 10 µ l de plasmídeo extraído e 60 µ l do tipo que da água. Misturar e transferir para uma cubeta de quartzo de 60 µ l ou usar 2 µ l diretamente em um Nanodrop.</li>
	<li>Medir os espectros de absorção entre 230 a 320 nm. Calcule a concentração de plasmídeo da amostra usando a seguinte fórmula: <img alt="Equation 1" src="/files/ftp_upload/56528/56528eq1v3.jpg"> onde D é o factor de diluição aplicado (neste caso é 70/10), Ax é absorção x nm e 50 é o fator de conversão de µ g/mL para dsDNA.</li>
	<li>Dilua as amostras para 9,1 ng / µ l.</li>
	<li>Transformar células competentes de Escherichia coli (e. coli) (por exemplo, XL10 Gold) seguindo a transformação Inoue do procedimento16 usando 5 µ l de amostra diluída.</li>
	<li>Contar o número de colônias sobre os pratos de Petri e observe a diluição. Calcular a colônia formando unidades (CFU) com a seguinte fórmula17: <img alt="Equation 2" src="/files/ftp_upload/56528/56528eq2v2.jpg"> onde D é o factor de diluição, aplicado desde a cultura inicial.</li>
	<li>Execute um teste-t não pareado.</li>
</ol>

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I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+DNA+using+a+visible+dye,+Nile+blue,+in+electrophoresed+gels.">Detection of DNA using a visible dye, Nile blue, in electrophoresed gels.</a> Anal. Biochem. 280 (2), 322-324 (2000).</li><li>Santill&#225;n-Torres, J. 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Os autores não têm nada para divulgar.

Um sensível e romance método de coloração de DNA com base na redução do NBT e o uso do SG, que foi apresentado neste artigo. O passo crítico neste protocolo é o tempo de incubação do gel em NBT-SG eu solução e a concentração de NBT. O tempo de coloração para gel de agarose é maior que para géis de acrilamida por causa da maior espessura de gel de agarose. Esse método não funciona bem na presença de SDS como NBT precipita-se em contacto com ele. Se o gel Obtém um fundo roxo, recomendamos que o gel de...

Com os avanços em bioquímica e biologia molecular, os estudos envolvendo DNA exigiram melhores técnicas para analisar o DNA. O primeiro método para coloração de DNA foi prata coloração, que é muito sensível, mas carece de seletividade e não permite a recuperação de amostra. Mais tarde, o desenvolvimento de coloração fluorescente de DNA permitiu a seletiva quantificação de DNA com a possibilidade de recuperação da amostra. Dentre os corantes fluorescentes primeiros usados para quantificação de DNA foi de brometo de etídio1, que é mutagênico2. No entanto, agora existem corantes fluorescentes melhorados mais seguros e mais sensíveis, tais como GelRed e SYBR Green eu (SG eu)3; Mas todos estes corantes fluorescentes exigem o uso de um transiluminador de (UV) ultravioleta ou fluorímetro.
Existem outras técnicas para coloração visivelmente o DNA, como o azul de metileno4 e violeta cristal5,6,7,8,9, mas todos estes sofrem de sensibilidade reduzida e seletividade. Os sais de tetrazólio são suscetíveis a redução de compostos orgânicos e quando isso acontece eles formam um insolúvel e colorida formazan precipitar10,11. Recentemente, foram mostrados para ligar ao DNA devido seus anéis de tetrazólio positivo12sais de tetrazólio bivalente.
Em uma recente publicação13, foi proposta uma nova técnica visível para manchar e quantificação de DNA em gel de poliacrilamida, usando a redução de um sal de tetrazólio bivalente chamado nitro azul tetrazólio (NBT) e corantes fluorescentes como brometo de etídio, GelRed, SYBR Green I e SYBR Gold. Esta reação funcionou na presença de luz azul ou luz solar e permitiu a recuperação da amostra com qualidade melhorada em comparação com o uso de SG eu com um transiluminador UV. O objetivo deste trabalho é fornecer um protocolo detalhado da técnica de coloração usando tetrazólio sais.

<table border="0" cellpadding="0" cellspacing="0" width="785">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Nitro blue tetrazolium</td>
			<td style="width:227px;">Gold Biotechnology</td>
			<td style="width:307px;">298-83-9</td>
			<td style="width:64px;">reagent used in the staining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">SYBR Green I 10000X</td>
			<td style="width:227px;">Thermo-Fisher</td>
			<td style="width:307px;">S7563</td>
			<td>reagent used in the staining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Gel extraction kit</td>
			<td style="width:227px;">QIAGEN</td>
			<td style="width:307px;">28704</td>
			<td>used to extract plasmid from the gel in integrity assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">DNA ladder</td>
			<td style="width:227px;">Thermo-Fisher</td>
			<td style="width:307px;">SM 1331</td>
			<td>used to make calibration curves and as reference to cut band in integrity assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Agar Agar</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;"></td>
			<td>used to make LB-ampicillin culture plates&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">sodium chloride</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">1064045000</td>
			<td>used to make LB-ampicillin culture plates&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">yeast extract</td>
			<td style="width:227px;">Becton, Dickinson and Company</td>
			<td style="width:307px;">212750</td>
			<td>used to make LB-ampicillin culture plates&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">peptone</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">72161000</td>
			<td>used to make LB-ampicillin culture plates&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">TEMED</td>
			<td style="width:227px;">AMRESCO</td>
			<td style="width:307px;">761</td>
			<td>used to make polyacrylamide gels</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">ammonium persulfate</td>
			<td style="width:227px;">Calbiochem</td>
			<td style="width:307px;">2310</td>
			<td>used to make polyacrylamide gels</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">acrylamide</td>
			<td style="width:227px;">invitrogen</td>
			<td style="width:307px;">15512-023</td>
			<td>used to make polyacrylamide gels</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">bisacrylamide</td>
			<td style="width:227px;">SIGMA</td>
			<td style="width:307px;">146072</td>
			<td>used to make polyacrylamide gels</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Tris-base</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">1083821000</td>
			<td>used to make TAE buffer and non-denaturing polyacrylamide gel tris-HCl buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">EDTA</td>
			<td style="width:227px;">J.T. Baker</td>
			<td style="width:307px;">8993-01</td>
			<td>used to make TAE buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Acetic acid</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">1,000,632,500</td>
			<td>used to make TAE buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">agarose</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">16802</td>
			<td>used to make TAE buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">spectrophotometer</td>
			<td style="width:227px;">Tecan</td>
			<td style="width:307px;">infinite 200 pro</td>
			<td>used to quantify DNA plasmid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">water purificatiom unit</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">Elix 100</td>
			<td>use to make water type 1 (ASTM) used in spectrophotometry and DNA dilutions\</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">distilled water</td>
			<td style="width:227px;"></td>
			<td style="width:307px;">-</td>
			<td>used in gels, culture medium, stainings and general solutions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">HCl</td>
			<td style="width:227px;">J.T. Baker</td>
			<td>9535-03</td>
			<td>used to make non-denaturing polyacrylamide gel tris-HCl buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">6X DNA loading dye</td>
			<td style="width:227px;">Thermo-Fisher</td>
			<td style="width:307px;">R0610</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">5 mm Blue LED</td>
			<td style="width:227px;">Jinyuan electronics</td>
			<td style="width:307px;">SP-021</td>
			<td>light source used in integrity assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">protoboard</td>
			<td style="width:227px;">KANDH</td>
			<td style="width:307px;">KH102</td>
			<td>light source support and circuit&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">9 V battery</td>
			<td style="width:227px;">Duracell</td>
			<td style="width:307px;">-</td>
			<td>used to power the LED&#39;s</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:187px;">horizontal electrophoresis chamber</td>
			<td style="width:227px;">Biorad</td>
			<td style="width:307px;">1704486EDU</td>
			<td>used to make and run agarose gel in integrity assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">vertical electrophoresis kit</td>
			<td style="width:227px;">Biorad</td>
			<td style="width:307px;">1658002EDU</td>
			<td>used to run polyacrylamide gels in calibration curves</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">power supply</td>
			<td style="width:227px;">Biorad</td>
			<td style="width:307px;">1645050&#160;</td>
			<td>used to power the electrophoresis</td>
		</tr>
	</tbody>
</table>

dna staining method based on formazan precipitation induced by blue light exposure

Métodos de coloração de DNA são muito importantes para a investigação biomédica. Nós projetamos um método simples que permite a visualização de DNA a olho nu, a formação de um precipitado colorido. Ele funciona através da imersão a acrilamida ou gel de agarose DNA em uma solução de 1 x (equivalente a 2,0 µM) SYBR Green eu (SG eu) e 0,20 mM nitro azul tetrazólio que produz um roxo precipitado de formazan quando expostos à luz solar ou especificamente a luz azul. Também, testes de recuperação de DNA foram realizados utilizando um plasmídeo resistente à ampicilina em um gel de agarose corado com nosso método. Obteve-se um maior número de colônias com nosso método do que com a tradicional mancha usando SG eu com iluminação ultravioleta. O método descrito é rápido, específico e não-tóxico para a deteção de DNA, permitindo a visualização de biomoléculas a "olho nu", sem um transiluminador e é barato e apropriado para uso em campo. Por estas razões, o nosso DNA novo método de coloração tem potenciais benefícios para a investigação e a indústria.

Um precipitado de formazan roxo aparece onde se encontra o DNA (verificado por espectrometria de massa13). No experimento, uma escada de DNA foi carregada para fazer uma curva de calibração (Figura 3). O protocolo funciona para gel de agarose e acrilamida, mas tem uma menor intensidade e leva mais tempo em agarose. No entanto, o uso de géis de agarose permite a recuperação da amostra usando um kit disponível comercialmente, e nã...

Watch this Scientific Journal Video about DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure at JoVE.com

DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

Este método permite coloração seletiva e quantificação de DNA em géis embebendo o gel em um SYBR Green eu / Nitro azul tetrazólio solução e então exposto à luz solar ou uma fonte de luz azul. Isto produz um precipitado visível e não requer quase nenhum equipamento, tornando-a ideal para uso em campo.

Método baseado na precipitação de Formazan induzida pela exposição à luz azul de coloração de DNA

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the ...

dna-staining-method-based-on-formazan-precipitation-induced-blue

Research

JoVE Journal

Biochemistry

9.4K visualizações.  Universidad de Chile. Este método permite coloração seletiva e quantificação de DNA em géis embebendo o gel em um SYBR Green eu / Nitro azul tetrazólio solução e então exposto à luz solar ou uma fonte de luz azul. Isto produz um precipitado visível e não requer quase nenhum equipamento, tornando-a ideal para uso em campo.

Vídeo: DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Resolver purificada por afinidade complexos de proteínas por Azul Native PAGE e Proteína Correlação Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Bioquímica

Real-time Tracking of DNA Fragment Separation by Smartphone

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and others. However, the traditional SGE protocol is quite tedious, and the experiment takes a long time. Moreover, the chemical consumption in SGE experiments is very high. This work proposes a simple method for the separation of DNA fragments based on an SGE chip. The chip is made by an engraving machine. Two plastic sheets are used for the excitation and emission wavelengths of the optical signal. The fluorescence signal of the DNA bands is collected by smartphone. To validate this method, 50, 100, and 1,000 bp DNA ladders were separated. The results demonstrate that a DNA ladder smaller than 5,000 bp can be resolved within 12 min and with high resolution when using this method, indicating that it is an ideal substitute for the traditional SGE method.

Traditional slab gel electrophoresis (SGE) experiments require a complicated apparatus and high chemical consumption. This work presents a protocol that describes a low-cost method to separate DNA fragments within a short timeframe.

Rastreamento em tempo real da separação de fragmentos de DNA por smartphone

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis can be characterized using an in vitro DNA synthesis assay followed by polyacrylamide gel electrophoresis. The goal of this assay is to quantify synthesis of both natural DNA and modified DNA (M-DNA). These approaches are particularly useful for resolving oligonucleotides with single nucleotide resolution, enabling observation of individual steps during enzymatic oligonucleotide synthesis. These methods have been applied to the evaluation of an array of biochemical and biophysical properties such as the measurement of steady-state rate constants of individual steps of DNA synthesis, the error rate of DNA synthesis, and DNA binding affinity. By using modified components including, but not limited to, modified nucleoside triphosphates (NTP), M-DNA, and/or mutant DNA polymerases, the relative utility of substrate-DNA polymerase pairs can be effectively evaluated. Here, we detail the assay itself, including the changes that must be made to accommodate nontraditional primer DNA labeling strategies such as near-infrared fluorescently labeled DNA. Additionally, we have detailed crucial technical steps for acrylamide gel pouring and running, which can often be technically challenging.

This protocol describes the characterization of DNA polymerase synthesis of modified DNA through observation of changes to near-infrared fluorescently labeled DNA using gel electrophoresis and gel imaging. Acrylamide gels are used for high resolution imaging of the separation of short nucleic acids, which migrate at different rates depending on size.

Ensaio da atividade de DNA polimerase usando infravermelho fluorescente etiquetado DNA visualizado pela electroforese do Gel do acrilamido

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Um método simples para High Throughput Screening químico em Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D2O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

Eletroquímico deteção de deutério cinética efeito isotópico no transporte de elétrons extracelular em Shewanella oneidensis MR-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

Análise de complexos de proteínas de membrana tilacoides por electroforese em Gel azul nativo

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A&#946;, which forms neurotoxic amyloid aggregates in Alzheimer&#39;s disease (AD), has been shown to interact with fibrinogen. This A&#946;-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A&#946;-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the A&#946;-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of A&#946; and A&#946;-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of A&#946;-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A&#946;-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified A&#946;-fibrinogen interaction inhibitor.

Presented here are two methods that can be used individually or in combination to analyze the effect of beta-amyloid on fibrin clot structure. Included is a protocol for creating an in vitro fibrin clot, followed by clot turbidity and scanning electron microscopy methods.

Análise de β-amiloide-induzida de anormalidades na estrutura do coágulo de fibrina por espectroscopia e microscopia eletrônica

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability.&#160;

Protein binding microarray (PBM) experiments combined with biochemical assays link the binding and catalytic properties of DNA primase, an enzyme that synthesizes RNA primers on template DNA. This method, designated as high-throughput primase profiling (HTPP), can be used to reveal DNA-binding patterns of a variety of enzymes.

Reconhecimento da seqüência do ADN pelo primase do ADN usando o perfilamento da primase da elevado-produção

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.

Visualizando dinâmica sadrindo de receptor estéta de células t usando microscopia de folha de luz de rede

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by F&#246;rster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.

This protocol describes an original setup combining spectral and fluorescence lifetime measurements to evaluate F&#246;rster resonance energy transfer (FRET) between rhodamine-based fluorescent probes and lignin polymer directly in thick plant sections.

Medindo interações entre sondas fluorescentes e lignina em seções de plantas por sFLIM Com base na autofluorescência nativa

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...