Nous tenons à remercier Michael Reth, Hermann Schagger, et Margarita Camacho-Carvajal d&#39;appui scientifique. Ce travail a été financé par de la Bildung FORSYS Bundesministerium für Forschung et (BMBF), par BIOSS de la Deutsche Forschungsgemeinschaft (DFG), et soutenu en partie par l&#39;Initiative d&#39;excellence des gouvernements fédéral allemand et de l&#39;Etat (CGC-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>

Dans cette étude, nous décrivons l&#39;analyse des PPM par BN-PAGE. Une approche 2D est utilisée d&#39;abord PPM séparés dans des conditions natives, puis de continuer à les subdiviser en leurs composants individuels par une deuxième dimension SDS-PAGE. Les échantillons sont préparés à partir de lysats cellulaires. Pour la solubilisation des PPM nombreuses, un détergent approprié est nécessaire, ce qui préserve la structure des complexes protéiques. Ici, nous utilisons 0,1% de Triton X-100. Toutefois, le détergent et sa concentration optimale appropriées doivent être déterminées empiriquement pour chaque PPM. En cas de Triton X-100, par exemple, il a été rapporté que les concentrations de détergent bas permettent l&#39;identification d&#39;une forme dimérique de la F 1 F 0-ATPase complexes (Arnold, Pfeiffer et al. 1998). Supérieur Triton X-100 concentrations, cependant, conduire à la dissociation du dimère et une augmentation correspondante de l&#39;monomère F 1 F 0-ATPase complexes. Ceci est en ligne avec l&#39;un de nos études antérieures, ont été, nous montrons que le complexe récepteur des cellules T multivalents (TCR) est conservée lorsque extrait avec de faibles concentrations de Brij 96, tandis que l&#39;utilisation de plus forte concentration de détergent ou d&#39;un autre appelé des résultats de la digitonine l&#39;extraction des monomères TCR (Schamel, Aréchaga et al. 2005). Détergents couramment utilisés qui peuvent être testés comprennent digitonine (0,5 à 1%), le Triton X-100 (0,1 à 0,5%), Brij 96 (0,1 à 0,5%), ou dodecylmaltoside (0,1 à 0,5%). Ces réactifs sont des détergents non ioniques, qui tendent à être les meilleurs pour la stabilité MPC. Soyez conscient que le contact avec SDS et d&#39;autres détergents puissants doivent être évités (Camacho-Carvajal, Wollscheid et al. 2004). Dialyse des lysats est nécessaire pour obtenir la séparation MPC dans un BN-gel (Camacho-Carvajal, Wollscheid et al 2004.); (Heiss, Junkes et al 2005).. Il semble que l&#39;ajustement de la concentration de sel ou de l&#39;élimination des impuretés de faible poids moléculaire est cruciale pour une grande résolution. Il est à noter que également des préparations de membrane et des PPM, qui ont été immunopurifié et plus tard sur élue à partir de l&#39;anticorps, sont adaptés pour BN-PAGE (Swamy, Siegers et al. 2006). Dans les deux cas, les échantillons n&#39;ont pas à être dialysé pour BN-PAGE de séparation, si lyse membrane ou élution est effectuée dans BN-tampon de lyse. Pour la séparation des protéines par BN-PAGE, le colorant bleu de Coomassie est nécessaire, qui se lie aux protéines non spécifique et les couvre de charges négatives. Ainsi, le bleu de Coomassie permet la mobilité électrophorétique des protéines vers la cathode à pH neutre (Schagger et Jagow 1991); (Schagger, Cramer et al, 1994).. Par ailleurs, le bleu de Coomassie empêche l&#39;agrégation des protéines dans le gel empilage durant l&#39;électrophorèse. Pour BN-PAGE, Coomassie G250 doit être utilisé au lieu du bleu de Coomassie R250 ou colloïdale bleus Coomassie. Avant de lancer une BN-gel, il est nécessaire de s&#39;assurer que le pourcentage du gel s&#39;adapte à la taille attendue de la MPC d&#39;intérêt. Préfabriqué BN-gels avec différents gradients et tampons appropriés sont disponibles commercialement auprès de Invitrogen (NativePAGE Novex Bis-Tris Gel System). Mais BN-gels peuvent également être préparés en utilisant un mélangeur de gradient avec une pompe persistaltic. Afin de garantir un gradient intactes, le liquide doit s&#39;écouler en permanence pendant la coulée et les bulles doivent être évités. Nous recommandons le chargement des dilutions des échantillons différents sur le gel, car la surcharge peut conduire à la précipitation des protéines au cours du procédé d&#39;électrophorèse. En outre, BN-gels doivent être exécutés à 4 ° C pour prévenir la dégradation des protéines et de garder intactes les PPM. Après BN-PAGE, la visualisation des PPM peut être réalisé par coloration bleu brillant de Coomassie, coloration à l&#39;argent ou d&#39;immunoblot. Bandes de protéines visualisés par coloration au bleu de Coomassie ou en argent sont adaptés à une analyse approfondie par spectrométrie de masse (Camacho-Carvajal, Wollscheid et al. 2004). En cas d&#39;immunoblot, les conditions de transfert optimal pour les PPM d&#39;intérêt doivent être déterminées empiriquement. Soyez conscient que le bleu de Coomassie est également transféré au cours d&#39;un buvard BN-gel. Par conséquent, le gel sera incolore après le transfert réussi, tandis que la membrane va exercer une couleur bleue. De plus, il est important de mentionner que chaque anticorps primaire, qui travaille pour la détection après SDS-PAGE, est applicable à immunoblot après BN-PAGE. Il peut arriver que les anticorps ne reconnaissent pas le MPC d&#39;intérêt parce que leur épitope est caché dans la conformation native des protéines. Pour surmonter ce problème, il est possible de dénaturer les protéines au sein du BN-gel avant le transfert en faisant bouillir le gel peu dans le tampon 1x échantillon de SDS. Dans notre exemple, nous n&#39;avons pas l&#39;objet de la BN-gel directement à la détection des bandes de protéines. Au lieu de cela, nous avons divisé le BN-PAGE, séparés par une seconde lysat dimensisur SDS-PAGE. Dans la deuxième dimension sur gel SDS, les protéines monomériques migrer à l&#39;intérieur d&#39;une diagonale hyperboliques due au gel de gradient dans le premier et le gel linéaire dans la deuxième dimension (Camacho-Carvajal, Wollscheid et al. 2004). Cela permet l&#39;identification facile des PPM, car elles sont localisées en dessous de cette diagonale hyperbolique. Sous-composants d&#39;un MPC distincts sont séparés dans une ligne verticale dans la deuxième dimension SDS-PAGE. Les composants qui sont des constituants des PPM dinstinct plusieurs peuvent être identifiés sur une ligne horizontale en fonction de la taille de la MPC. Cependant, il doit être considéré que les taches plusieurs protéines figurant dans une ligne verticale pourrait aussi faire partie de complexes distincts qui migrent à la même position dans BN-PAGE. La preuve définitive qu&#39;ils sont présents dans la même MPC peut être obtenue par un dosage à base d&#39;anticorps gel de quart de travail. Dans cet essai, lysat cellulaire est incubée avec un anticorps dirigé contre une protéine représentée par l&#39;un des endroits identifiés avant BN-PAGE. Il en résulte un décalage de tous les PPM qui contiennent cette protéine vers une masse moléculaire plus élevée dans la première dimension. D&#39;autres protéines qui sont aussi une partie de ces PPM va subir ce changement de complexes spécifiques et sont donc faciles à identifier dans la deuxième dimension SDS-gel. Non seulement la composition des PPM peuvent être analysés par BN-PAGE, mais aussi la détermination de leur stœchiométrie est possible (et Schamel Reth 2000); (Schamel 2001), (Swamy, Minguet et al 2007).. À cette fin, un test NAMOS (native à base d&#39;anticorps mobilité changement de dosage) peut être effectuée. Comme dans le dosage des anticorps gel à base de quart de travail, les lysats cellulaires sont incubés avec des anticorps monoclonaux sous-unité d&#39;anticorps spécifiques. Cela conduit à l&#39;induction de immunoshifts électrophorétique dans les gels BN-, qui permettent l&#39;inférence à partir de la mesure de l&#39;évolution de la stoechiométrie des PPM En guise de conclusion, BN-PAGE est approprié pour l&#39;identification des PPM et la détermination de leur taille, la composition, ainsi que l&#39;abondance relative. Exécutée comme une épreuve NAMOS, il offre également la possibilité de déterminer la stœchiométrie d&#39;une certaine MPC. Compte tenu de son applicabilité générale, cette technique est un outil très utile pour la caractérisation des PPM (Dekker, Müller et al 1996.); (Wittig et Schagger 2008); (Wagner, Rehling et al 2009.); (Wittig et Schagger 2009) .

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

Complexes multiprotéiques (PPM) jouent un rôle crucial dans la signalisation cellulaire, puisque la plupart des protéines peuvent être trouvés dans des complexes fonctionnels ou réglementaires avec d&#39;autres protéines (Sali, Glaeser et al. 2003). Ainsi, l&#39;étude des réseaux d&#39;interactions protéine-protéine requiert la caractérisation détaillée des PPM d&#39;acquérir une compréhension intégrative des fonctions des protéines et la réglementation. Pour l&#39;identification et l&#39;analyse, les PPM doivent être séparés dans des conditions natives. Dans cette vidéo, nous décrivons l&#39;analyse des PPM par le bleu électrophorèse en gel de polyacrylamide natif (BN-PAGE). BN-PAGE est une technique qui permet la séparation des PPM dans une conformation native avec une résolution supérieure à celle offerte par filtration sur gel ou d&#39;ultracentrifugation de densité de sucrose, et est donc utile pour déterminer la taille MPC, la composition et l&#39;abondance relative (Schagger et Jagow 1991) ; (Schagger, Cramer et al, 1994).. Par cette méthode, les protéines sont séparées selon leur taille et la forme hydrodynamique dans une matrice de polyacrylamide. Ici, nous démontrons l&#39;analyse des PPM du total des lysats cellulaires, soulignant que la dialyse lysat est l&#39;étape cruciale pour faire BN-PAGE applicables à ces prélèvements biologiques. En utilisant une combinaison de la dimension et la dimension BN-première seconde SDS-PAGE, nous montrons que les PPM séparés par BN-PAGE peut être subdivisée en leurs constituants individuels par SDS-PAGE. Visualisation des composants MPC en cas de séparation du gel est réalisée par immunoblot standard. À titre d&#39;exemple pour l&#39;analyse des PPM par BN-PAGE, nous avons choisi le 19S bien caractérisés eucaryotes, 20S, 26S et de protéasomes.

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

Dans cette vidéo, nous décrivons la caractérisation de complexes multiprotéiques (PPM) en bleu électrophorèse en gel de polyacrylamide natif (BN-PAGE). Dans une première dimension, dialysées lysats cellulaires sont séparés par BN-PAGE pour identifier les PPM individuelle. Dans une deuxième dimension SDS-PAGE, PPM d&#39;intérêt sont subdivisées afin d&#39;analyser leurs électeurs par immunoblot.

Bleu d&#39;électrophorèse sur gel de polyacrylamide natif (BN-PAGE) pour l&#39;analyse des complexes multiprotéiques de lysats cellulaires

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 vues.  University of Freiburg. Dans cette vidéo, nous décrivons la caractérisation de complexes multiprotéiques (PPM) en bleu électrophorèse en gel de polyacrylamide natif (BN-PAGE). Dans une première dimension, dialysées lysats cellulaires sont séparés par BN-PAGE pour identifier les PPM individuelle. Dans une deuxième dimension SDS-PAGE, PPM d'intérêt sont subdivisées afin d'analyser leurs électeurs par immunoblot.

Vidéo: 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.

Électrophorèse sur gel dénaturant Dégradé (DGGE)

Recherche

Biologie

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.

Le dépistage de l&#39;agrégation amyloïde par les semi-électrophorèse sur gel dénaturant Détergent-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.

Identification des complexes protéiques avec la protéomique quantitative chez 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.

Électrophorèse sur gel dénaturant Urée polyacrylamide (PAGE urée)

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 
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A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

Électrophorèse sur gel d&#39;agarose pour la séparation de fragments d&#39;ADN

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.

Une nouvelle approche pour l&#39;analyse comparative des complexes multiprotéiques Basé sur<sup&gt; 15</sup&gt; N Marquage métabolique et spectrométrie de masse quantitative

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.

La cartographie sur-AFM des propriétés élastiques de la paroi cellulaire: au tissu, cellulaire, et des résolutions Subcellular

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.

Mucine Agarose Gel Electrophoresis: Western Blot pour les glycoprotéines de haut poids moléculaire,


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.

Purification de Native Complexes pour l&#39;étude structurale utilisant une méthode de Tag Tandem Affinity

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

Bleu d'électrophorèse sur gel de polyacrylamide natif (BN-PAGE) pour l'analyse des complexes multiprotéiques de lysats cellulaires

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