Gostaríamos de agradecer ao laboratório Lee, da Universidade de Minnesota para fornecer o rato espinhal amostras de tecido espinhal que foram usados. Este trabalho foi financiado pelo Departamento de Defesa do ovário Cancer Research Program (OCRP) OC093424 a MB, pelo Fundo de Investigação e Comunidade Cancer Randy Shaver para MB e pelo Minnesota Ovarian Cancer Alliance (MOCA) para MB. Os financiadores não tiveram nenhum papel no desenho do estudo, coleta de dados e análise, decisão de publicar ou preparação do manuscrito.

1. Preparação de tampão de lise <ol><li> Dissolve-se a sacarose em água desionizada (Dl) para produzir uma solução stock a 2 M. Filtro de uma solução 2 M de sacarose utilizando um aspirador accionado 0,22 um fluoreto de polivinilideno (PVDF) de filtro. </li><li> Dissolve-se ditiotreitol (DTT) em água desionizada para produzir uma solução stock de 500 mM e armazenar sob condições anaeróbias. Dissolve-se cloreto de magnésio (MgCl2) em água desionizada para produzir uma solução stock de 100 mM. Dissolve-se trifosfato de adenosina (ATP), hidrato de dissódio em água desionizada para produzir uma solução stock de 50 mM. </li><li> Adicione-se uma solução de Tris a pH 7,4 através da dissolução de cloridrato de Trizma em água desionizada e ajustando o pH com hidróxido de sódio (NaOH). </li><li> Combinar porções das soluções de reserva em água Dl para fazer um tampão de lise com uma concentração de sacarose de 250 mM, uma concentração de 1 mM de DTT, uma concentração de MgCl2 5 mM, uma concentração de ATP de 1 mM e uma concentração de 50 mM Tris . Por exemplo, misturar 535,3 ul de Tris, 500 ul de MgCl2, 1,25 mL de sacarose, 20 l de DTT, 200 ul de ATP e 7,4947 ml de água desionizada para tornar 10 ml de tampão de lise. Isto pode ser utilizado para cerca de 20 experiências. </li></ol> 2. Células de cultura para o experimento <ol><li> Adicione meios de alíquotas de 30 ml de DMEM + 10% de soro fetal de bovino (FBS) para um tubo de 50 ml. </li><li> Transferência de células HeLa M17 ou congelados de armazenamento de nitrogênio líquido para um pequeno copo com água morna. </li><li> Transferir as células para o tubo de 50 ml. Primeiro, adicione alguns meios de comunicação para o frasco de celular. Em seguida, transferir as células ressuspensas dentro de 5 segundos de descongelamento. </li><li> Centrifugar a suspensão de células a 450 xg durante 5 minutos para se obter uma pelete de células. Adicionar 20 ml de mídia em um frasco T75. </li><li> Remova a mídia a partir das células usando sucção. Adicionar 5 ml de meio fresco para a pelete de células e ressuspender. </li><li> Transferir a suspensão de 5 ml de células para 20 ml de meio no frasco T75 e incubar a 37 ° C. Change os meios de comunicação a cada 3 dias. </li></ol> Colheita 3. celular <ol><li> Remova a mídia através de sucção, tendo o cuidado de não tocar nas células. </li><li> Lavam-se as células com 5 ml de solução salina tamponada com fosfato (PBS). Remover PBS utilizando sucção. </li><li> Adicionar 3 ml de tripsina-ácido etilenodiaminotetracético (EDTA) para o frasco T75 e incubar a 37 ° C durante 3 min. </li><li> Adicionar 6 ml de meio fresco ao frasco e voltar a suspender as células. </li><li> Transferir a suspensão para um tubo de 15 ml e centrifugar a 290 x g durante 5 minutos. </li><li> Remover o sobrenadante e adicionar 5 ml de PBS. Bata levemente o fundo do tubo para quebrar o sedimento. Centrifugar a 290 xg durante 5 min. </li><li> Repita o passo 3,6 três vezes. </li><li> Remover PBS e ressuspender as células em 1 ml de PBS fresco. Transferência para o tubo Eppendorf (1,5 ml). Gire a 290 g durante 5 min. </li></ol> 4. Lise Celular <ol><li> Medir o volume aproximado de o sedimento utilizando uma pipeta de umd pesam-se contas de vidro em uma massa: proporção em volume de 1: 1. Adicionar duas vezes o volume da pelota em tampão de lise. Nota: Adicionar o tampão antes de adicionar as contas de vidro. </li><li> Lisar as células no tubo de Eppendorf, no máximo de agitação durante 30 min a 4 ° C com vortex numa sala fria. </li><li> Centrifugar brevemente a 200 xg durante 30 seg para resolver os grânulos. Recolhe-se o sobrenadante. </li><li> Adicionar o mesmo volume de contas de vidro como antes para o sobrenadante. </li><li> Vortex, mais uma vez, no máximo de agitação durante 30 min a 4 ° C. </li><li> Centrifugar brevemente a 200 xg durante 30 segundos para se estabelecer as esferas de vidro. Recolhe-se o sobrenadante. </li><li> Centrifugar a 5.030 xg durante 5 minutos para remover os núcleos, membranas e as células intactas. Recolhe-se o sobrenadante, que é o lisado celular. </li></ol> A homogeneização 5. Tissue <ol><li> Faça uma massa: volume de 1: 9 solução de rato tecido do cordão espinhal de lise. Nota: Este método é aplicável a uma variedade de diferemamostras de tecido ENT. </li><li> Homogeneizar o tecido, utilizando uma configuração de nível 2 no homogeneizador durante 30 segundos para garantir que não existem partes restantes. </li><li> Girar a 5030 xg durante 3 minutos para remover os núcleos, membranas e as células intactas. </li><li> Recolhe-se o sobrenadante, que é o lisado. </li></ol> 6. Amostra Derivatizaï¿½o <ol><li> Realizar um ácido bicinconínico (BCA) em uma análise de placa de 96 alvéolos para determinar a concentração de proteína de lisado de tecido usando um colorimétrico leitor de placa de 96 poços, tal como especificado pelo protocolo do kit de Ensaio de Proteína Pierce BCA. </li><li> Trazer uma alíquota correspondente a 20 ug de proteína total de 50 ul usando (DI) de água desionizada. </li><li> Adicionar 2 mL de 1,35 M de sulfona HA-Ub-vinil (VS) para a solução e incubar durante 1 hora a 37 ° C. Isto resulta 20 ug de lisado a 50 nM de sonda na mistura de reacção final. Isto é, em grande excesso molar para assegurar a marcação dos dubs funcionais. </li><li> EUncubate da amostra em tampão de amostra de Laemmli para 5 min a 95 ° C. Para uma reacção de 52 ul de volume de utilização 26 ul de tampão de amostra 2x, 13 ul de tampão de amostra 4x ou 8,87 ul de tampão de amostra de 6x. </li><li> Arrefecer em gelo antes de carregar o gel. </li></ol> 7. Western Blot <ol><li> Carregar um de 12 poços de 4-20% de gel de Tris-glicina com 40 ul de amostra preparada </li><li> Submeter o gel a 95 V até a frente de íon atinge o fundo. </li><li> Realizar uma transferência durante a noite do gel para uma membrana de difluoreto de polivinilideno (PVDF). </li><li> Incubar a membrana no amido mancha preta e digitalizar o membane para obter uma imagem que mostra a quantidade de proteína em cada pista. </li><li> Destain a membrana e incubar em 5% de leite em PBS durante 1 h para bloquear. </li><li> Incubar a membrana em que o anticorpo anti-HA primária a uma concentração de 1: 10.000 em leite 5% em PBS, quer durante a noite a 4 ° C ou durante 4 horas à temperatura ambiente. </li><li> Realize 3 lavagens fou 5 minutos cada em PBS com 0,1% de Tween-20 em detergente. </li><li> Incubar a membrana de uma peroxidase de rábano rato a uma concentração de 1: 10.000 em leite 5% em PBS durante pelo menos 2 horas. </li><li> Realizar 3 lavagens durante 5 min cada em PBS com 0,1% de Tween-20 em detergente. </li><li> Realize uma lavagem por 5 min em PBS. </li><li> Incubar a membrana de um reagente de detecção quimioluminescente durante 10 min e detecção utilizando um detector de quimioluminescência. Use a configuração de exposição automática no detector. </li></ol>

<ol>
	<li>Ovaa, H., Kessler, B. M., Rol&#233;n, U., Galardy, P. J., Ploegh, H. L., Masucci, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-+based+ubiquitin-specific+(USP)+profiling+of+virus-infected+and+malignant+human+cells.">Activity- based ubiquitin-specific (USP) profiling of virus-infected and malignant human cells.</a> Proc Natl Acad Sci USA. 101 (8), 2253-2258 (2004).</li><li>Wilkinson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitination+and+deubiquitination:+Targeting+of+proteins+for+degradation+by+the+proteasome.">Ubiquitination and deubiquitination: Targeting of proteins for degradation by the proteasome.</a> Semin Cell Dev Biol. 11 (3), 141-148 (2000).</li><li>Ziad, M. E., Wilkinson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+proteolysis+by+human+deubiquitinating+enzymes.">Regulation of proteolysis by human deubiquitinating enzymes.</a> Biochim Biophys Acta. 1843 (1), 114-128 (2014).</li><li>Rol&#233;n, U., 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=Activity+Profiling+of+Deubiquitinating+Enzymes+in+Cervical+Carcinoma+Biopsies+and+Cell+Lines.">Activity Profiling of Deubiquitinating Enzymes in Cervical Carcinoma Biopsies and Cell Lines.</a> Mol Carcinog. 45 (4), 260-269 (2006).</li><li>Borodovsky, A., 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=Chemistry-Based+Functional+Proteomics+Reveals+Novel+Members+of+the+Deubiquitinating+Enzyme+Family.">Chemistry-Based Functional Proteomics Reveals Novel Members of the Deubiquitinating Enzyme Family.</a> Chem Biol. 9 (10), 1149-1159 (2002).</li><li>Andersson, F. L., 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=The+effect+of+Parkinson&#8217;s-disease-associated+mutations+on+the+deubiquitinating+enzyme+UCH-L1.">The effect of Parkinson&#8217;s-disease-associated mutations on the deubiquitinating enzyme UCH-L1.</a> J Mol Biol. 407 (2), 261-272 (2011).</li><li>Poon, W. W., 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=&#946;-Amyloid+(A&#946;)+oligomers+impair+brain-derived+neurotrophic+factor+retrograde+trafficking+by+down-regulating+ubiquitin+C-terminal+hydrolase+UCH-L1.">&#946;-Amyloid (A&#946;) oligomers impair brain-derived neurotrophic factor retrograde trafficking by down-regulating ubiquitin C-terminal hydrolase UCH-L1.</a> J Biol Chem. 288 (23), 16937-16948 (2013).</li><li>Nijman, S. M., 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=The+deubiquitinating+enzyme+USP1+regulates+the+Fanconi+anemia+pathway.">The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway.</a> Mol Cell. 17 (3), 331-339 (2005).</li><li>Stevenson, L. F., Sparks, A., Allende-Vega, N., Xirodimas, D. P., Lane, D. P., Saville, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+deubiquitinating+enzyme+USP2a+regulates+the+p53+pathway+by+targeting+Mdm2.">The deubiquitinating enzyme USP2a regulates the p53 pathway by targeting Mdm2.</a> EMBO J. 26 (4), 976-986 (2007).</li><li>Coughlin, K., 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=Small-molecule+RA-9+inhibits+proteasome-associated+DUBs+and+ovarian+cancer+in+vitro+and+in+vivo+via+exacerbating+unfolded+protein+responses.">Small-molecule RA-9 inhibits proteasome-associated DUBs and ovarian cancer in vitro and in vivo via exacerbating unfolded protein responses.</a> Clin Cancer Res. 20 (12), 3174-3186 (2014).</li><li>Colla, E., 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=Endoplasmic+Reticulum+Stress+Is+Important+for+the+Manifestations+of+&#945;-Synucleinopathy+In.">Endoplasmic Reticulum Stress Is Important for the Manifestations of &#945;-Synucleinopathy In.</a> Vivo. J Neurosci. 32 (10), 3306-3320 (2012).</li></ol>

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Desde ubiquitinação é uma actividade celular fundamental, a compreensão dos mecanismos de regulação poderia ser a chave para desenterrar os processos de doença e patologia. O uso de HA etiquetado local dirigida sondas ativas Ub-derivadas aqui relatados fornece um método fácil, mas altamente aplicável para estudar a degradação de proteínas mediada por Ub. Este método é mais rápido e menos dispendioso do que o estudo de cada uma das dobragens individualmente. Neste método, a l...

O sistema ubiquitina-proteassoma (UPS) serve como uma das principais vias de degradação na célula de mamífero. Substratos ligados à degradação no proteassoma são covalentemente marcados com polímeros de ubiquitina (Ub) 1. Antes do substrato alvo entra no proteassoma para a degradação, a etiqueta de poli-ubiquitina deve ser removido. Uma classe de enzimas conhecidas como deubiquitinating enzimas (Dubs) é responsável pela remoção e reciclagem de moléculas de ubiquitina 2. Prevê-se a partir do genoma humano que há quase uma centena de dobragens de trabalho na célula 3. Com um grande número de dubs que controlam processos celulares Ub mediadas tal, estudando estas enzimas apresenta um desafio desde técnicas de mRNA não dão informação sobre a actividade e transferência de western só dá informações sobre os níveis de expressão. A utilização da gripe hemaglutinina (HA) com etiquetas, Ub-derivado local dirigida sondas activas permite uma modificação covalenteficação das dobragens funcionais e, por conseguinte, dá uma visualização directa da actividade destas enzimas sobre um western blot 4. As sondas têm um grupo reactivo tiol C-terminal que serve como um substrato para o suicídio resíduo cisteína local activo 5. Com estas sondas, é possível estudar a expressão e a actividade potencial de muitos dobragens em ambos os estados patológicos e fisiológicos da célula. Alterações na actividade DUB têm sido implicados numa variedade de condições patológicas tais como doença de Parkinson, doença de Alzheimer, anemia e vários cancros 6-10. Esta técnica fornece uma ferramenta poderosa para o estudo da doença. No presente trabalho, mostramos a aplicação desta técnica em células HeLa e M17 que foram ligado usando esferas de vidro. Além disso, destacamos como usar esse método em amostras de tecido de rato medula espinhal. A informação obtida a partir desta técnica pode ser usada como um ponto de partida para a identificaçãoalvos terapêuticos, bem como o estabelecimento de modelos para o estudo de diferentes condições de doença. A verdadeira utilidade desta técnica reside na sua capacidade de fornecer informações sobre vários Dubs em um único ensaio.

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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PowerGen 125 ( Homogenizer)</td>
			<td style="width:113px;">Fischer Scientific</td>
			<td style="width:119px;">14-261-02P</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vacuum-driven filters .22&#181;M&#160;</td>
			<td style="width:113px;"></td>
			<td style="width:119px;">BPV2210</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass beads, acid washed &#8804;106&#181;m (~140 U.S. sieve)</td>
			<td style="width:113px;">Sigma-aldrich</td>
			<td style="width:119px;">G4649-10G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sucrose</td>
			<td style="width:113px;">Fischer Scientific</td>
			<td style="width:119px;">S6-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DL-Dithiothreitol</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:119px;">D0632</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium Chloride</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8266</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Adenosine 5&#39;-triphosphate disodium salt hydrate</td>
			<td style="width:113px;">Sigma-aldrich</td>
			<td style="width:119px;">A26209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trizma hydrochloride&#160;</td>
			<td style="width:113px;">Sigma-aldrich</td>
			<td style="width:119px;">T3253</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:113px;">Life technologies</td>
			<td style="width:119px;">11965-092</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phosphate Buffered Saline</td>
			<td style="width:113px;">Life technologies</td>
			<td style="width:119px;">10010-023</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tissue culture flask 75cm2 w/ filter cup 250 ml 120/ cs</td>
			<td style="width:113px;">Cellstar</td>
			<td style="width:119px;">T-3001-2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">0.05% Trypsin-EDTA (1X)</td>
			<td style="width:113px;">Life technologies</td>
			<td style="width:119px;">25300-054</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HI FBS</td>
			<td style="width:113px;">Life technologies</td>
			<td style="width:119px;">16140-071</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Monoclonal Anti-HA antibody produced in mouse</td>
			<td style="width:113px;">Sigma-aldrich</td>
			<td style="width:119px;">H9658</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ubiquitin vinyl sulfone (HA-tag)</td>
			<td style="width:113px;">Enzo Life Sciences</td>
			<td style="width:119px;">BML-UW0155-0025</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Laemmli&#39;s SDS-Sample Buffer (4X, reducing)</td>
			<td style="width:113px;">Boston BioProducts</td>
			<td style="width:119px;">BP-110R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pierce BCA Protein Assay Kit</td>
			<td style="width:113px;">ThermoScientific</td>
			<td align="right" style="width:119px;">23225</td>
			<td></td>
		</tr>
	</tbody>
</table>

method for measuring the activity of deubiquitinating enzymes in cell lines and tissue samples

O sistema ubiquitina-proteassoma tem sido recentemente implicado em diversas patologias, incluindo as doenças neurodegenerativas e cancro. À luz disto, técnicas para estudar o mecanismo de regulação deste sistema são essenciais para elucidar os processos celulares e moleculares das doenças acima mencionadas. A utilização de sondas derivado de hemaglutinina ubiquitina descritos neste documento serve como uma ferramenta valiosa para o estudo deste sistema. Este artigo detalha um método que permite ao usuário realizar ensaios que dão uma visualização direta do deubiquitinating atividade enzimática. Deubiquitinating controlar a degradação proteossómica e compartilhar homologia funcional em seus sítios ativos, o que permite ao usuário investigar a atividade de várias enzimas em um ensaio. Lysates são obtidos através de suave rompimento celular mecânica e incubadas com o site ativo sondas dirigidas. Enzimas funcionais são marcados com as sondas enquanto enzimas inativos permanecem não ligados. Por prazoNing este ensaio, o utilizador obtém informações sobre a actividade, tanto a expressão e potencial de várias enzimas deubiquitinating de uma maneira fácil e rápida. O método actual é significativamente mais eficiente do que a utilização de anticorpos específicos para os preditos cem enzimas deubiquitinating na célula humana.

As células M17 e HeLa em cultura foram colhidos usando o método detalhado na (de colheita celular 3.) protocolo e do tecido da medula espinal do rato foi obtido. O sedimento de células / tecido da medula espinal foi colocada num tubo com o tampão de lise descrito na secção de preparação do reagente. As pelotas de células foram lisadas usando pérolas de vidro (Figura 1A) e amostras de tecido de ratinho medula espinal foram homogeneizados usando o homogeneizador (Figura 1B). Ap?...

Watch this Scientific Journal Video about Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples at JoVE.com

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

O protocolo atual detalha um método para medir a atividade das enzimas funcionalmente homóloga deubiquitinating. Sondas especializados modificar covalentemente o enzima e permitir a detecção. Este método tem o potencial para identificar novos alvos terapêuticos.

Método para medir a actividade de enzimas deubiquitinating em linhas celulares e amostras de tecidos

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, ...

method-for-measuring-activity-deubiquitinating-enzymes-cell-lines

Research

JoVE Journal

Biology

9.5K visualizações.  University of Minnesota.  O protocolo atual detalha um método para medir a atividade das enzimas funcionalmente homóloga deubiquitinating. Sondas especializados modificar covalentemente o enzima e permitir a detecção. Este método tem o potencial para identificar novos alvos terapêuticos. 

Vídeo: Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

Actina Ensaio Co Sedimentação; para a Análise da proteína de ligação a F-Actina

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Biologia

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &amp;#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

Quantificação de γH2AX Focos em amostras de tecido

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

Um ensaio para medir a atividade do Escherichia coli Induzível Lisina Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Do-It-Yourself Dispositivo de Recuperação de amostras criopreservadas cair acidentalmente em Tanques de armazenamento criogênico

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Measuring In Vitro ATPase Activity for Enzymatic Characterization

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization. Proteins hydrolyze ATP in a reaction that results in inorganic phosphate release, and the amount of phosphate liberated is then quantitated using a colorimetric assay. This highly adaptable protocol can be adjusted to measure ATPase activity in kinetic or endpoint assays. A representative protocol is provided here based on the activity and requirements of EpsE, the AAA+ ATPase involved in Type II Secretion in the bacterium Vibrio cholerae. The amount of purified protein needed to measure activity, length of the assay and the timing and number of sampling intervals, buffer and salt composition, temperature, co-factors, stimulants (if any), etc. may vary from those described here, and thus some optimization may be necessary. This protocol provides a basic framework for characterizing ATPases and can be performed quickly and easily adjusted as necessary.

Measuring In Vitro ATPase Activity for Enzymatic Characterization

We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.

Medindo<em&gt; In Vitro</em&gt; Actividade ATPase para enzimática Caracterização

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate ...

Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility with high-throughput formatting. This protocol describes the use of three different methods for measuring cell proliferation in vitro including conventional hemocytometer counting chamber, a luminescence-based assay that utilizes the change in the metabolic activity of viable cells as a measure of the relative number of cells, and a multi-mode cell imager that measures cell number using a counting algorithm. Each method presents its own advantages and disadvantages for the measurement of cell proliferation, including time, cost and high-throughput compatibility. This protocol demonstrates that each method could accurately measure cell proliferation over time, and was sensitive to detect growth at differing cellular densities. Additionally, measurement of cell proliferation using a cell imager was able to provide further information such as morphology, confluence and allowed for a continual monitoring of cell proliferation over time. In conclusion, each method is capable of measuring cell proliferation, but the chosen method is user-dependent.

This protocol describes the use of three different methods for analyzing cell proliferation in breast cancer cell lines. This includes the use of conventional cell counting, luminescence-based cell viability, and cell counting through the use of a cell imager. Each offers advantages for the reproducible measurement of cell proliferation.

Comparação de três métodos diferentes de determinação da proliferação celular em linhas celulares de cancro da mama

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility ...

Measuring Lactase Enzymatic Activity in the Teaching Lab

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 &#176;C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

Medindo a atividade enzimática de Lactase no laboratório de ensino

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Hybrid-Cut: Um Método Seccionamento melhorado para amostras de tecidos de plantas recalcitrante

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called the auditory bulla houses the ossicles, whereas the auditory capsule encloses the inner ear, namely the cochlea and semicircular canals. Murine ossicles are essential for hearing and thus of great interest to researchers in the field of otolaryngology, but their metabolism, development, and evolution are highly relevant to other fields. Altered bone metabolism can affect hearing function in adult mice, and various gene-deficient mice show changes in morphogenesis of auditory ossicles in utero. Although murine auditory ossicles are tiny, their manipulation is feasible if one understands their anatomical orientation and 3D structure. Here, we describe how to dissect the auditory bulla and capsule of postnatal mice and then isolate individual ossicles by removing part of the bulla. We also discuss how to embed the bulla and capsule in different orientations to generate paraffin or frozen sections suitable for preparation of longitudinal, horizontal, or frontal sections of the malleus. Finally, we enumerate anatomical differences between mouse and human auditory ossicles. These methods would be useful in analyzing pathological, developmental and evolutionary aspects of auditory ossicles and the middle ear in mice.

We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis.

Dissecção da Auditivo Bulla em ratos pós-natais: Isolamento dos ossos do ouvido médio e da análise histológica

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-Clear: Um método de compensação por imagem tridimensional do tecido adiposo tecido

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...