Agradecemos a Michael Reth, Hermann Schägger, e Margarita Camacho-Carvajal ao apoio científico. Este trabalho foi financiado pela Forsys do fuer Bundesministerium Bildung e Forschung (BMBF), pela BIOS do Deutsche Forschungsgemeinschaft (DFG), e apoiado em parte pela Iniciativa de Excelência dos Governos Federal e Estadual alemães (GSC-4, Spemann Graduate School ).

<ol>
	<li>Arnold, I., Pfeiffer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+mitochondrial+F1F0-ATP+synthase+exists+as+a+dimer:+identification+of+three+dimer-specific+subunits.">Yeast mitochondrial F1F0-ATP synthase exists as a dimer: identification of three dimer-specific subunits.</a> EMBO J. 17 (24), 7170-7178 (1998).</li><li>Camacho-Carvajal, M. M., Wollscheid, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+Blue+native/SDS+gel+electrophoresis+of+multi-protein+complexes+from+whole+cellular+lysates:+a+proteomics+approach.">Two-dimensional Blue native/SDS gel electrophoresis of multi-protein complexes from whole cellular lysates: a proteomics approach.</a> Mol. Cell. Proteomics. 3 (2), 176-182 (2004).</li><li>Heiss, K., Junkes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subproteomic+analysis+of+metal-interacting+proteins+in+human+B+cells.">Subproteomic analysis of metal-interacting proteins in human B cells.</a> Proteomics. 5 (14), 3614-3622 (2005).</li><li>Sali, A., Glaeser, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+words+to+literature+in+structural+proteomics.">From words to literature in structural proteomics.</a> Nature. 422, 216-225 (2003).</li><li>Schägger, H., Cramer, W. A. <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+molecular+masses+and+oligomeric+states+of+protein+complexes+by+blue+native+electrophoresis+and+isolation+of+membrane+protein+complexes+by+two-dimensional+native+electrophoresis.">Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis.</a> Anal. Biochem. 217 (2), 220-230 (1994).</li><li>Sch&#228;gger, H., von Jagow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+electrophoresis+for+isolation+of+membrane+protein+complexes+in+enzymatically+active+form.">Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form.</a> Anal. Biochem. 199 (2), 223-231 (1991).</li><li>Schamel, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotinylation+of+protein+complexes+may+lead+to+aggregation+as+well+as+to+loss+of+subunits+as+revealed+by+Blue+Native+PAGE.">Biotinylation of protein complexes may lead to aggregation as well as to loss of subunits as revealed by Blue Native PAGE.</a> J Immunol Methods. 252 (1-2), 171-174 (2001).</li><li>Schamel, W. W., Arechaga, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coexistence+of+multivalent+and+monovalent+TCRs+explains+high+sensitivity+and+wide+range+of+response.">Coexistence of multivalent and monovalent TCRs explains high sensitivity and wide range of response.</a> J. Exp. Med. 202, 493-503 (2005).</li><li>Schamel, W. W., Reth, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monomeric+and+oligomeric+complexes+of+the+B+cell+antigen+receptor.">Monomeric and oligomeric complexes of the B cell antigen receptor.</a> Immunity. 13 (1), 5-14 (2000).</li><li>Swamy, M., Minguet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+native+antibody-based+mobility-shift+technique+(NAMOS-assay)+to+determine+the+stoichiometry+of+multiprotein+complexes.">A native antibody-based mobility-shift technique (NAMOS-assay) to determine the stoichiometry of multiprotein complexes.</a> J Immunol Methods. 324 (1-2), 74-83 (2007).</li><li>Swamy, M., Siegers, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+polyacrylamide+gel+electrophoresis+(BN-PAGE)+for+the+identification+and+analysis+of+multiprotein+complexes.">Blue native polyacrylamide gel electrophoresis (BN-PAGE) for the identification and analysis of multiprotein complexes.</a> Sci STKE. 2006 (345), 4-4 (2006).</li><li>Wittig, I., Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+and+applications+of+blue-native+and+clear-native+electrophoresis.">Features and applications of blue-native and clear-native electrophoresis.</a> Proteomics. 8, 3974-3990 (2008).</li></ol>

Não há conflitos de interesse declarados.

Neste estudo, descrevemos a análise do PPM pelo BN-PAGE. Uma abordagem 2D é usada para separar primeiro PPM em condições nativas e, depois, subdividir-los em seus componentes individuais, uma segunda dimensão SDS-PAGE. As amostras são preparadas a partir de lisados ​​celulares. Para a solubilização do PPM muitos, um detergente apropriado é necessário, que preserva a estrutura dos complexos de proteína. Aqui, usamos 0,1% Triton X-100. No entanto, o detergente ideal e adequada a sua concentração tem que ser determinado empiricamente para cada MPC. Em caso de Triton X-100, por exemplo, foi relatado que as concentrações de detergente permitir a identificação de uma forma dimérica da F 1 F 0-ATPase complexos (Arnold, Pfeiffer et al. 1998). Maior Triton X-100 concentrações, no entanto, levam à dissociação do dímero e um aumento correspondente do monomérica F 1 F 0-ATPase complexo. Isto está de acordo com um dos nossos estudos anteriores, nós mostramos que foram o complexo receptor de células T multivalentes (TCR) é preservada quando extraído com baixas concentrações de Brij 96, enquanto que o uso de maior concentração ou de outro detergente chamado resultados digitonina em a extração de monomérica TCR (Schamel, Aréchaga et al. 2005). Detergentes comumente usados ​​que podem ser testados incluem digitonina (0,5 a 1%), Triton X-100 (0,1 a 0,5%), Brij 96 (0,1 a 0,5%), ou dodecylmaltoside (0,1 a 0,5%). Estes reagentes são os detergentes não-iônico, que tendem a ser melhor para a estabilidade MPC. Esteja ciente de que o contato com SDS e outros detergentes fortes devem ser evitados (Camacho-Carvajal, Wollscheid et al. 2004). Diálise do lisados ​​é necessário para alcançar MPC separação em um BN-gel (Camacho-Carvajal, Wollscheid et al 2004).; (Heiss, Junkes et al 2005).. Parece que o ajuste da concentração de sal ou a remoção de impurezas de baixo peso molecular é crucial em alta resolução. Vale ressaltar que também preparações de membrana e PPM, que têm sido immunopurified e, posteriormente, eluída da anticorpos, são adequados para BN-PAGE (Swamy, Siegers et al. 2006). Em ambos os casos, as amostras não precisam ser dialisado para BN-PAGE separação, se lise de membranas ou de eluição é realizada em BN lise-buffer. Para a separação de proteínas por BN-PAGE, o corante Coomassie blue é necessário, que se liga a proteínas não específica e cobre-os com cargas negativas. Assim, Coomassie blue permite a mobilidade eletroforética de proteínas para o catodo de pH neutro (Schägger e von Jagow 1991); (Schägger, Cramer et al 1994).. Além disso, Coomassie blue previne a agregação de proteínas no gel de empilhamento durante a eletroforese. Para BN-PAGE, Coomassie G250 tem que ser usado em vez de azul Coomassie R250 ou blues Coomassie coloidal. Antes de executar um BN-gel, é necessário para garantir que a percentagem de gel se adapta ao tamanho esperado do MPC de interesse. Pré-moldado BN-géis com gradientes diferentes e buffers adequados estão disponíveis comercialmente a partir Invitrogen (NativePAGE Novex Bis-Tris Sistema Gel). Mas BN-gel também pode ser preparado usando um misturador de gradiente, juntamente com uma bomba persistaltic. Para garantir um gradiente intactos, o líquido deve fluir constantemente durante o vazamento e bolhas devem ser evitadas. Recomendamos o carregamento de diferentes diluições de amostras no gel, pois a sobrecarga pode levar à precipitação de proteínas durante o processo de eletroforese. Além disso, BN-gel deve ser executado a 4 ° C para prevenir a degradação de proteínas e para manter o PPM intacta. Depois BN-PAGE visualização, de PPM pode ser alcançado por Coomassie coloração azul brilhante, coloração pela prata ou immunoblotting. Bandas de proteínas visualizados através da coloração Coomassie ou prata são adequados para posterior análise por espectrometria de massa (Camacho-Carvajal, Wollscheid et al. 2004). Em caso de immunoblotting, as condições de transferência ideal para o PPM de juros têm de ser determinadas empiricamente. Estar ciente de que Coomassie azul também é transferida durante blotting de um BN-gel. Portanto, o gel vai ser incolor após a transferência bem-sucedida, ao passo que a membrana irá exercer uma cor azul. Além disso, é importante mencionar que não todos os anticorpos primários, que trabalha para detecção após SDS-PAGE, é aplicável a immunoblotting sobre BN-PAGE. Pode acontecer que os anticorpos não reconhecem o MPC de interesse porque o seu epítopo está escondido na conformação nativa das proteínas. Para ultrapassar este problema, é possível para desnaturar as proteínas dentro da BN-gel antes da transferência fervendo o gel em breve no 1x tampão de amostra SDS. No nosso exemplo, não submeta o BN-gel diretamente para a detecção de bandas de proteínas. Em vez disso, nós ainda dividiu o BN-PAGE-lisado separados por um segundo dimensiem SDS-PAGE. Na segunda dimensão SDS-gel, as proteínas monoméricas migrar dentro de uma diagonal hiperbólica devido ao gradiente de gel no primeiro e no gel linear na segunda dimensão (Camacho-Carvajal, Wollscheid et al. 2004). Isto permite a fácil identificação de PPM, uma vez que estão localizados abaixo dessa diagonal hiperbólica. Subcomponentes de um MPC distintos são separados em uma linha vertical na segunda dimensão SDS-PAGE. Componentes que são constituintes do PPM dinstinct vários podem ser identificados em uma linha horizontal de acordo com o tamanho do MPC. No entanto, ele tem que se considerar que vários spots de proteínas que aparecem em uma linha vertical também poderia ser parte de complexos separados que migram na mesma posição em BN-PAGE. A prova final de que eles estão presentes no MPC mesmo pode ser obtido por um ensaio de alteração baseados em anticorpos gel. Neste ensaio, lisado celular é incubada com um anticorpo contra uma proteína representado por um dos pontos identificados antes da BN-PAGE. Isso resulta em uma mudança de todos os PPM que contêm essa proteína para uma maior massa molecular na primeira dimensão. Outras proteínas que são também uma parte destes PPM vai sofrer essa mudança complexos específicos e por isso são fáceis de identificar na segunda dimensão SDS-gel. Não só a composição do PPM podem ser analisados ​​por BN-PAGE, mas também a determinação de sua estequiometria é possível (e Schamel Reth 2000); (Schamel 2001), (Swamy, Minguet et al 2007).. Para este fim, um ensaio Namos (nativo baseadas em anticorpos, ensaio de mobilidade shift) pode ser realizada. Como no ensaio de mudança baseadas em anticorpos, gel, os lisados ​​celulares são incubados com anticorpos monoclonais subunidade anticorpos específicos. Isto leva à indução de immunoshifts eletroforética em gel-BN, que permitem a inferência da extensão da mudança na estequiometria do PPM Em conlusion, BN-PAGE é adequado para a identificação de PPM ea determinação de sua composição, tamanho, bem como abundância relativa. Realizada como um ensaio Namos, ela também oferece a possibilidade de determinar a estequiometria de uma MPC certos. Dada a sua aplicabilidade geral, esta técnica é uma ferramenta muito útil para a caracterização de PPM (Dekker, Müller et al 1996).; (Wittig e Schagger 2008); (Wagner, Rehling et al 2009.); (Wittig e Schägger 2009) .

blue native polyacrylamide gel electrophoresis (bn-page) for analysis of multiprotein complexes from cellular lysates

Complexos multiprotein (PPM) desempenham um papel crucial na sinalização celular, já que a maioria das proteínas podem ser encontradas em complexos funcionais ou de regulamentação com outras proteínas (Sali, Glaeser et al. 2003). Assim, o estudo das redes de interação proteína-proteína requer a caracterização detalhada do PPM para ganhar uma compreensão integrativa da função da proteína e regulação. Para a identificação e análise, PPM devem ser separados em condições nativas. Neste vídeo, descrevemos a análise do PPM por eletroforese em gel de poliacrilamida nativo azul (BN-PAGE). BN-PAGE é uma técnica que permite a separação de PPM em uma conformação nativa com uma resolução maior que o oferecido por filtração em gel ou ultracentrifugação de densidade de sacarose, e é, portanto, útil para determinar MPC tamanho, composição e abundância relativa (Schägger e von Jagow 1991) ; (Schägger, Cramer et al 1994).. Por este método, as proteínas são separadas de acordo com seu tamanho e forma hidrodinâmica em uma matriz de poliacrilamida. Aqui, demonstramos a análise do PPM do total lisados ​​celulares, apontando que a diálise lisado é o passo crucial para fazer BN-PAGE aplicável a estas amostras biológicas. Usando uma combinação de primeira dimensão BN e segunda dimensão SDS-PAGE, mostramos que PPM separados por BN-PAGE pode ser subdividida em seus componentes individuais por SDS-PAGE. Visualização dos componentes MPC sobre separação gel é realizada por immunoblotting padrão. Como um exemplo para MPC análise por BN-PAGE, que escolheu o 19S bem caracterizados eucarióticas, 20S, 26S e proteasomas.

Watch this Scientific Journal Video about Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates at JoVE.com

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

Neste vídeo, descrevemos a caracterização de complexos multiprotein (PPM) por eletroforese em gel de poliacrilamida nativo azul (BN-PAGE). Em uma primeira dimensão, dialisadas lisados ​​celulares são separados por BN-PAGE para identificar PPM individual. Em uma segunda dimensão SDS-PAGE, PPM de interesse são subdivididos para analisar os seus constituintes por 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 ...

blue-native-polyacrylamide-gel-electrophoresis-bn-page-for-analysis

Research

JoVE Journal

Biology

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

130.8K visualizações.  University of Freiburg. Neste vídeo, descrevemos a caracterização de complexos multiprotein (PPM) por eletroforese em gel de poliacrilamida nativo azul (BN-PAGE). Em uma primeira dimensão, dialisadas lisados ​​celulares são separados por BN-PAGE para identificar PPM individual. Em uma segunda dimensão SDS-PAGE, PPM de interesse são subdivididos para analisar os seus constituintes por immunoblotting.

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

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

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

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

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 ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Identificação de complexos de proteínas com proteômica quantitativa em S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

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 ...

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 
&#160;

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 ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

Uma Nova Abordagem para a Análise Comparativa de Complexos Multiproteicos Baseado em<sup&gt; 15</sup&gt; N Metabólica Labeling e quantitativa Espectrometria de Massa

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

Mapeamento baseado em AFM das propriedades elásticas de paredes celulares: em tecido, os celulares, e as Resoluções Subcelulares

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

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 ...

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree of compositional heterogeneity of a complex do not usually impede progress in conducting such studies. In contrast, a complex intended for structural characterization should be purified in a state that is both compositionally and structurally homogeneous as well as at a higher concentration than required for proteomics. Recently, there have been significant advances in the application of electron microscopy for structure determination of large macromolecular complexes. This has heightened interest in approaches to purify native complexes of sufficient quality and quantity for structural determination by electron microscopy. The Tandem Affinity Purification (TAP) method has been optimized to extract and purify an 18-subunit, ~ 0.8 MDa ribonucleoprotein assembly from budding yeast (Saccharomyces cerevisiae) suitable for negative stain and electron cryo microscopy. Herein is detailed the modifications made to the TAP method, the rationale for making these changes, and the approaches taken to assay for a compositionally and structurally homogeneous complex.

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

Purificação de Native Complexos de estudo estrutural usando um método Tag Tandem Affinity

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree ...

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

Resumo

Explore mais vídeos

PLAYLIST

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

Resumo

Explore mais vídeos

PLAYLIST

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