Agradecemos a Simon Alberti para obter ajuda com o desenvolvimento deste protocolo. Este trabalho foi suportado por uma concessão do National Institutes of Health (GM25874), um Howard Hughes Medical Institute Investigatorship (para SL), e da National Science Foundation bolsa de formação predoctoral (para RH).

<html> Parte 1: Preparação do gel <ol><li> Montar a bandeja de fundição gel. Equipamento padrão para eletroforese de DNA horizontal pode ser usada. Para um grande número de amostras, nós preparamos a 20 cm x 24 cm da laje com até quatro de 50 pentes bem. Certifique-se que a laje não tem arranhões, pois estas podem distorcer a imagem blot. </li><li> Criar uma solução de agarose 1,5% (média ou alta força de gel, EEO baixo) em 1X TAE. O volume deve ser suficiente para submergir completamente os dentes do pente - você vai querer carregar amostra, tanto quanto possível para maximizar a detecção. Microondas a mistura até que a agarose é completamente dissolvido. </li><li> Rapidamente adicionar SDS a 0,1% a partir de um estoque de 10%. Agite para misturar. Se alguns agarose solidifica durante esta etapa, dissolver novamente usando uma placa quente e ter cuidado para evitar a ferver. </li><li> Despeje a solução no tabuleiro casting. Use um pente para ajuntar qualquer bolhas, pois mais tarde poderão interferir com a transferência. </li><li> Depois de gel tem, retire os pentes e colocar o gel para o tanque de gel. Submergir completamente o gel em 1X TAE contendo 0,1% SDS. </li></ol> Parte 2: Preparando-se amostras <ol><li> Para high-throughput análise de lisados ​​de levedura, começamos com 2 ml-culturas cultivadas durante a noite com agitação rápida em 96 poços blocos. Neste caso, cada cultura é superexpressão de uma proteína de interesse. Ao analisar proteínas baixa abundância, maiores volumes de cultura deve ser utilizado </li><li> Células colheita por centrifugação a 2000 RCF por 5 min à temperatura ambiente. </li><li> Ressuspender as células em água e centrifugar novamente. </li><li> Ressuspender em 1 ml de solução spheroplasting. Incubar por aproximadamente 30 min a 30 ° C (você pode confirmar a eficiência spheroplasting por microscopia). </li><li> Centrifugar a 800 rcf durante 5 min à temperatura ambiente. Remover completamente o sobrenadante. </li><li> Ressuspender esferoplastos peletizadas em 100 ml tampão de lise. </li><li> Cubra o bloco com fita e vortex em alta velocidade por 2 min. </li><li> Pellet restos celulares em 4000 RCF por 2 min. </li><li> Remova cuidadosamente o sobrenadante para um novo recipiente, por exemplo, uma placa de 96 poços PCR. </li><li> Se desejar, determinar a concentração de proteína do lisados. </li><li> Adicionar tampão de amostra 4X às amostras para gerar lisados ​​contendo 1X tampão de amostra. Incubar por 5 min à temperatura ambiente. </li><li> Carga gel. Se desejar, salvar metade do volume de amostra e ferva por SDS-PAGE análise. A fim de monitorar a taxa de transferência mais tarde, incluem um pré-manchada marcador SDS-PAGE. Além disso, um marcador de peso molecular composta por proteínas muito grandes (por exemplo, frango peitoral extrato) pode ser usado para estimar os tamanhos dos complexos resolvido. </li><li> Executado em baixa tensão (≤ 3 comprimento do gel V / cm) até a frente de corante atinge ~ 1 cm do final do gel. Isso vai demorar várias horas. É importante que o gel de manter a calma, caso contrário, a difusão de proteínas de baixo peso molecular (por exemplo, monômeros) pode limitar a sua detecção. </li></ol> Parte 3: Transferência <ol><li> Corte um pedaço de nitrocelulose para as mesmas dimensões do gel. </li><li> Corte de 20 peças de GB004 e GB002 8 pedaços de papel absorvente, para as mesmas dimensões do gel. Corte uma peça adicional de GB004 para ser usado como um pavio; torná-lo cerca de 20 centímetros mais largo que o gel. </li><li> Mergulhe a nitrocelulose, pavio, e 4 pedaços de GB002 em 1X TBS. </li><li> Em um recipiente de plástico, montar uma pilha de papéis da seguinte forma: 20 peças de GB004 seca, em seguida, 4 pedaços de GB002 seca, em seguida, um pedaço de pré-molhada GB002. Lay a nitrocelulose no topo desta pilha. </li><li> Lavar o gel na bandeja de fundição brevemente em água para remover excesso de tampão de corrida. Então, com cuidado começar a deslizar o gel da bandeja na pilha. Enquanto desliza o gel da bandeja, extinguir a membrana com TBS, se necessário. O buffer extra ajuda a evitar bolhas de ficar preso sob o gel. A pipeta de transferência funciona bem para esta finalidade. </li><li> Depois que o gel foi movido à pilha, analisar cuidadosamente as bolhas. Caso existam, levante a borda do gel e reaplicar tampão até que as bolhas podem ser trabalhados. </li><li> Coloque os restantes três pré-molhada GB002 peças em cima do gel. Garantir o contato completo entre todas as camadas rolando uma pipeta firmemente na parte superior da pilha. </li><li> Flanco a pilha de transferência com duas bandejas contendo elevados TBS. Drape o pavio pré-molhada em toda a pilha de tal forma que uma das extremidades do pavio está submerso em TBS. </li><li> Cobrir a pilha de transferência montado com uma bandeja de plástico adicionais rolamento de peso extra (por exemplo, uma garrafa de 500 ml de água). </li><li> Permitir a transferência para prosseguir para um mínimo de três horas, ou durante a noite. </li><li> Após a transferência, a membrana pode ser processado por padrão Western blotting. </li></ol> Solução Spheroplasting 1,2 M D-sorbitol 0,5 mM MgCl 2 20 mM Tris, pH 7.5 BME 50 mM (adicionar fresco) 0,5 mg / ml Zymolyase 100T (adicionar fresco) coration: underline; &quot;Tampão de Lise&gt; 100 mM Tris 7,5 50 mM NaCl 10 mM BME (adicionar fresco) inibidores da protease (adicionar fresco) Tampão de amostra 4X 2X TAE Glicerol 20% 8% SDS azul de bromofenol a preferência

<ol>
	<li>Kryndushkin, D. S., Alexandrov, I. M., Ter-Avanesyan, M. D., Kushnirov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+[PSI+]+prion+aggregates+are+formed+by+small+Sup35+polymers+fragmented+by+Hsp104.">Yeast [PSI+] prion aggregates are formed by small Sup35 polymers fragmented by Hsp104.</a> J. Biol. Chem. 278, 49636-49643 (2003).</li><li>Alexandrov, I. M., Vishnevskaya, A. B., Ter-Avanesyan, M. D., Kushnirov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appearance+and+Propagation+of+Polyglutamine-based+Amyloids+in+Yeast:+TYROSINE+RESIDUES+ENABLE+POLYMER+FRAGMENTATION.">Appearance and Propagation of Polyglutamine-based Amyloids in Yeast: TYROSINE RESIDUES ENABLE POLYMER FRAGMENTATION.</a> J. Biol. Chem. 283, 15185-15192 (2008).</li><li>Allen, K. D., 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=Hsp70+chaperones+as+modulators+of+prion+life+cycle:+novel+effects+of+Ssa+and+Ssb+on+the+Saccharomyces+cerevisiae+prion+[PSI+].">Hsp70 chaperones as modulators of prion life cycle: novel effects of Ssa and Ssb on the Saccharomyces cerevisiae prion [PSI+].</a> Genetics. 169, 1227-1242 (2005).</li><li>Aron, R., Higurashi, T., Sahi, C., Craig, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=J-protein+co-chaperone+Sis1+required+for+generation+of+[RNQ+]+seeds+necessary+for+prion+propagation..">J-protein co-chaperone Sis1 required for generation of [RNQ+] seeds necessary for prion propagation..</a> The EMBO journal. 26, 3794-3803 (2007).</li><li>Bagriantsev, S. N., Kushnirov, V. V., Liebman, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+amyloid+aggregates+using+agarose+gel+electrophoresis.">Analysis of amyloid aggregates using agarose gel electrophoresis.</a> Methods in Enzymology. 412, 33-48 (2006).</li><li>Borchsenius, A. S., Muller, S., Newnam, G. P., Inge-Vechtomov, S. G., Chernoff, Y. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+variant+maintained+only+at+high+levels+of+the+Hsp104+disaggregase.">Prion variant maintained only at high levels of the Hsp104 disaggregase.</a> Current Genetics. 49, 21-29 (2006).</li><li>Salnikova, A. B., Kryndushkin, D. S., Smirnov, V. N., Kushnirov, V. V., Ter-Avanesyan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonsense+suppression+in+yeast+cells+overproducing+Sup35+(eRF3)+is+caused+by+its+non-heritable+amyloids.">Nonsense suppression in yeast cells overproducing Sup35 (eRF3) is caused by its non-heritable amyloids.</a> J. Biol. Chem. 280, 8808-8812 (2005).</li><li>Tank, E. M., Harris, D. A., Desai, A. A., True, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+protein+repeat+expansion+results+in+increased+aggregation+and+reveals+phenotypic+variability.">Prion protein repeat expansion results in increased aggregation and reveals phenotypic variability.</a> Mol. Cell. Biol. 27, 5445-5455 (2007).</li><li>Douglas, P. 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=Chaperone-dependent+amyloid+assembly+protects+cells+from+prion+toxicity.">Chaperone-dependent amyloid assembly protects cells from prion toxicity.</a> Proc. Natl. Acad. Sci. USA. 105, 7206-7211 (2008).</li><li>Nagy, B., Costello, R., Csako, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Downward+blotting+of+proteins+in+a+model+based+on+apolipoprotein(a)+phenotyping.">Downward blotting of proteins in a model based on apolipoprotein(a) phenotyping.</a> Analytical Biochemistry. 231, 40-45 (1995).</li></ol>

SDD-AGE foi primeiramente relatada por Kryndushkin et al. 1, para estudar SDS-resistentes complexos do [PSI +] prion em levedura, e desde então tem encontrado uso generalizado estudar tanto prion e príon não-agregados 2-9. No entanto, a transferência das proteínas para uma membrana de eletroforese seguinte em um gel de agarose é problemático, e pode resultar em uma imagem distorcida blot 5. Além disso, a técnica submersa eletrobloting mais comumente utilizado apresenta limitações práticas para o tamanho do gel e, assim, o número de amostras que podem ser processados. Temos abordado estes problemas através do emprego de transferência capilar para baixo 10, um procedimento simples, que utiliza uma pilha de papéis secos blotting para transferir proteínas do gel para uma membrana de nitrocelulose. Transferência capilar evita a distorção e permite géis grande para ser processado facilmente. Há algumas coisas a considerar antes de usar SDD AGE. Para amostras de crude (por exemplo, lisados), imunodetecção de proteínas específicas é necessária. SDD-AGE não totalmente desnaturar a complexos de proteínas de interesse, de modo a proteína (s) a ser detectado deve ostentar uma etiqueta de epítopo fora da região amiloidogênicas. Lisados ​​geralmente podem ser preparados como se fossem para um normal SDS-PAGE, com duas diferenças importantes. Em primeiro lugar, o cuidado maior deve ser tomado para evitar a degradação por proteólise. As condições de desnaturação parcialmente usados ​​aqui não são suficientes para inativar proteases, e também pode fazer proteínas-alvo mais suscetível à proteólise. Use um coquetel inibidor de protease total em pelo menos duas vezes a concentração recomendada. Segundo, o aquecimento das amostras deve ser evitado. Se um controle de monômero de todos os negativos é desejada, por exemplo, para confirmar que de alto peso molecular de espécies não são devido a modificações covalentes, a incubação de 10 minutos a 95 ° C pode ser usada, que vai restaurar a maioria dos amilóides à proteína monomérica.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>zymolyase 100T</td>
<td>&nbsp;</td>
<td>Seikagaku America</td>
<td>120493-1</td>
<td>&nbsp;</td>
<tr>
<td>Halt Protease Inhibitor Cocktail</td>
<td>&nbsp;</td>
<td>Thermo Scientific </td>
<td>78429</td>
<td>&nbsp;</td>
<tr>
<td>Hybond-C extra nitrocellulose</td>
<td>&nbsp;</td>
<td>Amersham</td>
<td>RPN303E</td>
<td>&nbsp;</td>
<tr>
<td>GB004 blotting paper</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>10427926</td>
<td>&nbsp;</td>
<tr>
<td>GB002 (3MM Chr) blotting paper</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>3030-917</td>
<td>&nbsp;</td>
<tr>
<td>Agarose (UltraPure)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15510-027</td>
<td>&nbsp;</td>		</tbody>
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		</div>

screening for amyloid aggregation by semi-denaturing detergent-agarose gel electrophoresis

Agregação amilóide está associada com patologias diversas proteínas misfolding e subjaz as propriedades de príons infecciosos, que são auto-conformationally templating proteínas que são pensados ​​para ter papéis benéficos em organismos inferiores. Amilóides foram extremamente difíceis de estudar devido à sua insolubilidade e heterogeneidade estrutural. No entanto, a resolução de polímeros amilóide com base no tamanho e insolubilidade detergente tem sido possível graças ao Semi-desnaturação Detergente-Eletroforese em Gel de Agarose (SDD AGE). Esta técnica é encontrar amplo uso para a detecção e caracterização de variantes amilóide conformacional. Aqui, nós demonstramos uma adaptação dessa técnica que facilita seu uso em aplicações em larga escala, tais como telas para prions romance e outras proteínas amiloidogênicas. O método SDD-AGE novos usos de transferência capilar para maior confiabilidade e facilidade de uso, e permite que qualquer gel dimensionados para ser acomodados. Assim, um grande número de amostras, preparadas a partir de células ou proteínas purificadas, podem ser processadas simultaneamente para a presença de SDS-conformistas de proteínas insolúveis marcados.

Watch this Scientific Journal Video about Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis at JoVE.com

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

SDD-AGE é uma técnica útil para a detecção e caracterização de amilóide como polímeros nas células. Aqui nós demonstramos uma adaptação que torna esta técnica passível de aplicações em larga escala.

Triagem para Agregação de amilóide por Semi-desnaturação Eletroforese Gel Detergente-Agarose

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

screening-for-amyloid-aggregation-semi-denaturing-detergent-agarose

Research

JoVE Journal

Biology

26.8K visualizações.  Whitehead Institute for Biomedical Research. SDD-AGE é uma técnica útil para a detecção e caracterização de amilóide como polímeros nas células. Aqui nós demonstramos uma adaptação que torna esta técnica passível de aplicações em larga escala.

Vídeo: Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis DGGE

Okay.Hi, my name is Celeste Peterson and I'm a fellow at Harvard University studying microbial bio film communities. So I am profiling the bacterial communities by looking at their 60 N sequence and how this sequence changes over time in space. And to do that, I'm using DDGE.This is not a normal acry of my well, it needs to be kept at 60 degrees when it's run and the buffer needs to be pumped continuously. So we're gonna warm up the buffer for about an hour until it's at 60 degrees, and then we can run the towel. Now I'm gonna wash this plate with dilute uc solution, peeling around for impurities, andone, light that up.Anything like that can cause regularities. It is important to wipe off all the water. So when I assemble the gel, I'll put that chip on the outside of the gel.So the gel will be in between these two pieces of glass and the chip will be on the outside and it won't affect the gel in between. So now I'm gonna assemble the gel in these clamps and then I squeeze them together and tighten by turning to the right. As with any gel, it's important to make sure that the bottom is flush.To prevent making, we now turn the gel around and put it in here to get sealed with the gasket. So it's important when you do this that the gel, that the plates are actually lined up with the gasket here. So these knobs on the side then go in and you press down to type the seal.So this is a 30 mil syringe. And this part and this part come with the bio red kit and it just on like this. And that's just to get rid of the excess water in the tubes.So put the tubing onto the gel to load the gel. First you put an adapter into the tubing and then a needle look with that small adapter and put it right in the center of the gel just so it's fairly below the bottom glass. Then this wire adapter goes into the long tubing and it's going to connect to equal syringes containing the high and the low density solution.So these are pre-prepared solutions. The blue one, I've added a dye here that just indicates that this is the most denaturing solution and this is the least fitting train solution, the low density one. And they have everything in them.And now I'm gonna to make, gonna be Actually I twice as much the week. And once you've got the tempt, you have about five to seven minutes to fill the gel before the colon rises. And now we fill the syringe juice with the solution.So I'm gonna fill it with about 15 the solution. And then to get rid of any air bubbles, I'll turn it upside down and squeeze gently until that top air bubble is gone. Now this little screw here is gonna go into this groove.This adapter is gonna line up with this clamp here, and it's gonna get C clamped in very tightly. And then this tubing is gonna attach to the syringe. And the same thing is done on the other side with the high density matrix where it's time to deliver.And so I'm gonna be turning this wheel in a steady clockwise motion. And first the blue solution is gonna go out first because that's the the most in nature, and it's gonna form the bottom part of the gel. And then after a little while, the clear solution will start filling in the gel and the Gradient will be formed.One moment is the gently on an angle to And as soon as you finish pouring the gel to wash out your tubes immediately because they will polymerize Going, It's the group there. And then press down. So this little, And on the other side, I'll do the same thing.Okay.Hi, my name is Celeste Peterson and I'm a fellow at Harvard University studying microbial bio communities. So I am profiling the bacterial communities by looking at their 60 F sequence and how the sequence changes over time in space. And to do that, I'm using DDGE.So DGE is Dena gel electrophoresis. And basically the idea is that there's a gradient and the DNA, as it runs down the gradient gets de natured. And so different 16 species will stop at different points along the gradient.And so it ends up being a fingerprint of the different sexiness species in the community. So the goal of my experiment is to see who is in the community and how that's changing over time and space. And I can run the sexiness DDD gel and then I can cut out the bands and identify the species that are represented in each community.So one of the biggest issues when, when doing A DDG gel is to figure out the gradient that's the best. And so that is just something that needs to be played around with and figuring out for a particular community which type of gradient. So the one I'm using today is 35 to 55%urea.One of the big problems that can happen with this technique is that smears come up in in the leans. And I found for that, for whatever reason, one of the best things to do is to run the gels for a really long time and then the smears disappear. Well, today I'm just using it for general bacterial communities, but this can also be used for a specific family of bacteria to see how those change over time, and those tend to be very clean gels.You can also look at mutations in single genes with DDGE.

Eletroforese em Gel de gradiente desnaturante (DGGE)

Biologia

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method1. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 &deg;C during the gel run allows for the separation of unstructured DNA or RNA molecules.
In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.
In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol 1,2.

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.

Desnaturação Uréia Eletroforese em Gel de Poliacrilamida (PAGE Urea)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

Eletroforese em Gel de Poliacrilamida azul Nativo (BN-PAGE) para Análise de Complexos Multiproteicos de Lisados ​​Celular

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Pouring and Running a Protein Gel by reusing Commercial Cassettes

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry and molecular biology researchers1-4. For laboratories that perform daily analyses of proteins, the cost of commercially available polyacrylamide gels (&tilde;$10/gel) can be considerable over time. To mitigate this cost, some researchers prepare their own polyacrylamide gels. Traditional methods of pouring these gels typically utilize specialized equipment and glass gel plates that can be expensive and preclude pouring many gels and storing them for future use. Furthermore, handling of glass plates during cleaning or gel pouring can result in accidental breakage creating a safety hazard, which may preclude their use in undergraduate laboratory classes. Our protocol demonstrates how to pour multiple protein gels simultaneously by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment. In addition, plastic gel cassettes are extremely resistant to breakage, which makes them ideal for undergraduate laboratory classrooms.

Our protocol demonstrates how to pour multiple protein gels at a time by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment.

Derramando e executando um gel de proteína através da reutilização Cassetes comerciais

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry ...

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel&#39;s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is &#34;biased reptation&#34;, whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 
 
1. Understand the mechanism by which DNA fragments are separated within a gel matrix 
2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 
3. Identify an agarose solution of appropriate concentration for their needs 
4. Prepare an agarose gel for electrophoresis of DNA samples 
5. Set up the gel electrophoresis apparatus and power supply 
6. Select an appropriate voltage for the separation of DNA fragments 
7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 
8. Determine the sizes of separated DNA fragments 
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A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

Electroforese em gel de agarose para separação dos fragmentos de DNA

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo Rastreamento 4-Dimensional de tronco hematopoéticas e células progenitoras em adultos mouse na calota craniana de Medula Óssea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Rastreio de alto rendimento para moduladores químicos de Genes Post-transcriptionally Regulamentados

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

Mucina Agarose Gel Eletroforese: Western Blotting para glicoproteínas alta peso molecular


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

Métodos de estudo mudanças na agregação de proteínas inerente com a idade em Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

Microscopia confocal revela agregação de receptores de superfície celular através de espectroscopia de correlação de imagem

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...