Nous tenons à remercier tous les membres du laboratoire Dinman, y compris Ashton Belew, Karen Jack, Sharmishtha Musalga, Sergey Sulima, Shivani Reddy, et Michael Rhodin pour leur aide et leur participation à ce projet. Ce travail a été soutenu par des subventions à l&#39;AM de l&#39;American Heart Association (AHA 0630163N) et à partir du JDD National Institutes of Health (5R01 GM058859-12). JAL a été soutenue par un réinvestissement américains et Recovery Act de 2009 supplément à la subvention des parents (3R01GM058859-11S1).

1. Préparation d&#39;une cystéine chargé Sulfolink de résine <ol><li> Préparer Sulfolink résine (Pierce) à température ambiante. </li><li> Placer un total de 10 ml d&#39;une suspension de 50% de gel de couplage Sulfolink tel que fourni par le fabricant dans deux flacons de 10 ml en plastique, avec 5 ml distribué dans chaque flacon. </li><li> Centrifuger brièvement flacons à 850 xg et retirez soigneusement surnageants. </li><li> Lavez les trois gel de fois dans le tampon de couplage (50 mM Tris, pH 8,5, EDTA 5 mM) par remise en suspension des billes dans 5 ml, centrifuger brièvement à 850 xg et élimination du surnageant à la pipette. </li><li> Ajoutez cinq ml d&#39;une solution 50 mM de L-cystéine dans le tampon de couplage à chaque tube et mélanger le lisier pendant 1 heure à 25 ° C. </li><li> Enlever le résidu L-cystéine par un lavage comme décrit dans l&#39;étape 1.4 ci-dessus. </li><li> Reprendre le gel dans 10 ml de tampon de liaison [20 mM HEPES, pH 7,6, 5 mM de Mg (OAc) 2, 60 mM de NH 4 Cl, 2 mM DTT], et équilibrer la résine par lavage dans 10 ml de tampon de liaison3 fois comme décrit dans l&#39;étape 1.4. Décanter la résine dans une colonne de 10 ml par centrifugation, casquette et conservés à 4 ° C. La capacité de la résine pour la liaison à l&#39;ARN est ~ 20 A 260 unités par ml. </li></ol> 2. Purification chromatographique des ribosomes en utilisant une cystéine chargé Sulfolink de résine Remarque: Si vous travaillez avec une souche où l&#39;activité de la protéase se révèle être un problème, cocktails inhibition des protéases peut être utilisé dans tous les tampons suivants. <ol><li> Maintenir tous les composants sur la glace et préparer toutes les solutions utilisant l&#39;eau sans RNase et la filtration stérile. </li><li> Cultivez les cellules de levure nuit à la mi-phase log (DO 595 = 0,6 - 1,2) dans les médias appropriés. Cellules par centrifugation, et laver les cellules 1 gramme dans le tampon de liaison et centrifuger à 3700 xg pendant 5 minutes à 4 ° C. </li><li> Resuspendre les cellules dans 1 ml de tampon de liaison à un volume total approximatif de 2 ml, et de perturber l&#39;aide d&#39;un volume égal à 0,5 mm de perles de verre réfrigéré à 4 ° C uchanter une Biospec Mini-perles batteur (Bartlesville, OK). Les échantillons peuvent être divisés en plusieurs tubes, au besoin. </li><li> Retirer les cellules ininterrompue, les organites, et les débris cellulaires par centrifugation à 30000 xg pendant 30 minutes dans une ultracentrifugeuse Beckman-Coulter Optima Max E (Fullerton, CA). </li><li> Immédiatement avant utilisation, granulés de la résine pendant une minute à 1000 xg pour éliminer la solution de stockage. Deux ml de résine est utilisée pour chaque gramme de cellules. </li><li> Retirer surnageants lysat cellulaire de la vrille 30000 xg (S30), en prenant soin de minimiser la contamination soit de la fraction lipidique au sommet ou les débris cellulaires au fond des tubes, et placer directement dans la boue Sulfolink facturés. La couche lipidique peut être évité soit par enlèvement ou en poussant à partir de la pipette, après la pointe a traversé dans le surnageant. </li><li> Incuber le mélange S30/Sulfolink sur la glace sans mélanger pendant 15 minutes. </li><li> Placer les colonnes dans 15 ml tubes coniques et centrifuger à 1000 xg pendantr une minute. </li><li> Placer les fractions accréditives retour dans les colonnes, mélanger à la main, et incuber pendant encore 15 minutes sur la glace. </li><li> Placer les colonnes dans 15 ml tubes coniques et centrifuger à 1000 xg pendant une minute. Jeter les actions accréditives. </li><li> Cap colonnes et ajouter 5 ml de tampon de liaison. Mélanger à la main jusqu&#39;à colonnes resuspendues, enlever les bouchons et centrifuger la centrifugeuse colonnes à 1000 xg pendant une minute. Répétez ce protocole de lavage deux fois plus. </li><li> Ajouter 1,5 ml de tampon d&#39;élution (20 mM HEPES-KOH, pH 7,6, 10 mM Mg (OAc) 2, 500 mM de KCl, 2 mM de DTT, 0,5 mg / ml d&#39;héparine) pour chaque colonne. Mélanger boues à la main, et incuber sur glace pendant 2 minutes </li><li> Placer les colonnes dans de nouveaux tubes coniques de 15 ml et centrifuger à 1000 xg pendant 1 minute. Recueillir l&#39;éluat. Répétez l&#39;étape d&#39;élution fois de plus pour que les derniers volumes de l&#39;échantillon combiné sont ~ 3 ml. Résine utilisée peut être lavée avec un tampon d&#39;élution sans héparine et suivie par équilibration avec Buffe contraignanter, et stockés dans 10 volumes ml de tampon de liaison à 4 ° C pour les réutiliser plus tard. </li></ol> 3. Traitement de puromycine Note: Une méthode alternative pour enlever la contamination des espèces ARNt est de passer d&#39;un glucose riche pour les médias afin de promouvoir le glucose appauvri les eaux de ruissellement des ribosomes. Toutefois, cela ne change l&#39;état métabolique des cellules de levure, ce qui pourrait affecter la fonction du ribosome et la concentration. Pour dépouiller les ribosomes endogènes du peptidyl-ARNt, un traitement avec puromycine est effectuée. <ol><li> Neutraliser à pH 7,5 100 mM solution de puromycine en ajoutant goutte à goutte de KOH 1M à température ambiante. </li><li> Ajouter la solution neutralisée à la puromycine ~ 1 mM concentration finale dans l&#39;éluat. </li><li> Ajouter 100 mM GTP à une concentration finale de 1 mM. </li><li> Incuber pendant 30 minutes à 30 ° C. </li></ol> 4. Purification des ribosomes par sédimentation grâce à des coussins de glycérol <ol><li> Placer un mlde tampon coussin (20 mM HEPES, pH 7,6, 10 mM Mg (OAc) 2, 500 mM de KCl, 2 mM de DTT, glycérol 25%) dans un tube de 4 volumes ultracentrifugeuse ml en polycarbonate. </li><li> Délicatement la couche puromycine traités fractions d&#39;élution sur le dessus du coussin, et des échantillons de centrifugation à 100.000 xg nuit à 4 ° C. </li><li> Retirer les tubes de l&#39;rotor de la centrifugeuse, et aspirer surnageants. </li><li> Laver des pastilles contenant des ribosomes purifié deux fois avec 1 ml de tampon de coussin. </li><li> Resuspendre ribosomes dans 100 ul de tampon coussin par des perturbations douces en utilisant une tige de verre. </li><li> Après les perturbations, couvrir les tubes avec du parafilm et agiter à une vitesse modérée dans un tourbillon chambre froide pendant une heure. </li><li> Transférer le contenu dans un tube à centrifuger, centrifuger pendant 5 minutes à vitesse maximale à 4 ° C, et de supprimer les surnageants dans des tubes frais. </li><li> Répétez les étapes 04/01 au 04/07 avec 2 ml de tampon coussin et, sauf que ul 100 de stockage tampon (50 mM HEPES-KOH pH 7,6, 50 mM de NH 4 Cl, 5 mM de Mg(OAc) 2, DTT 1 mM, glycérol 25%) est utilisé dans les étapes 4.4 et 4.5 au lieu de tampon coussin. </li><li> Quantifier les ribosomes purifiée par spectrophotométrie (1 A 260 = 20 pmoles de ribosomes de levure), et conserver à -80 ° C. Les résultats typiques d&#39;1 gramme de levure sont de 300 à 400 pmoles de ribosomes. </li></ol> 5. Les résultats représentatifs: Un exemple d&#39;espèces d&#39;ARN extrait de chacune des trois grandes étapes de ce protocole est montré dans la figure 2. Alors que les ARNr sont les principales espèces présentes dans les lysats cellulaires totaux (T), ces contiennent également un grand nombre d&#39;espèces d&#39;ARN d&#39;autres. Les ribosomes purifiée à partir de la colonne Sulfolink (SL) contiennent également une grande quantité d&#39;ARNt en raison de la forte affinité du lit de la colonne pour ces espèces ainsi. Le traitement de cette fraction avec des résultats puromycine dans l&#39;hydrolyse des peptides issus de la peptidyl-ARNt, et favorise la dissociation de ces espèces de ribosomes. La puromycine échantillons traités (Pm) l&#39;absence de co-purifying espèces ARNt, et représentent donc des ribosomes complètement pur. Comme indiqué précédemment 8, ces ribosomes sont très intact et biochimiquement active, ce qui les rend idéales pour les substrats détaillée des analyses fonctionnelles et structurelles. <img src="/files/ftp_upload/3214/3214fig1.jpg" alt="Figure 1"/> Figure 1. Organigramme méthode. Purification chromatographique des ribosomes utilisant la cystéine liée sulfolink résine est représenté. <img src="/files/ftp_upload/3214/3214fig2.jpg" alt="Figure 2"/> Figure 2. Représentant l&#39;analyse des préparations ribosome. Espèces d&#39;ARN total ont été extraites à partir de lysats cellulaires totaux (T), Sulfolink ribosomes purifié (SL) et purifiée ribosomes Sulfolink ensuite traité par la puromycine et sédimenté à travers un coussin de glycérol (Pm). Lane M représente la taille des marqueurs ARN. Bandes représentant ARNr 25S et 18S, ainsi que les ARNt et d&#39;autres espèces d&#39;ARN sont indiqués. Tous lesvoies ont été chargées avec 2 ug d&#39;ARN.

<ol>
	<li>Palade, G. E., Siekevitz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+microsomes;+an+integrated+morphological+and+biochemical+study.">Liver microsomes; an integrated morphological and biochemical study.</a> J. Biophys. Biochem. Cytol. 2, 171-200 (1956).</li><li>Fabry, M., Kalvoda, L., Rychlik, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophobic+interactions+of+Escherichia+coli+ribosomes.">Hydrophobic interactions of Escherichia coli ribosomes.</a> Biochim. Biophys. Acta. 652, 139-150 (1981).</li><li>Jelenc, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+purification+of+highly+active+ribosomes+from+Escherichia+coli.">Rapid purification of highly active ribosomes from Escherichia coli.</a> Anal. Biochem. 105, 369-374 (1980).</li><li>Le goffic, F., Moreau, N., Chevelot, L., Langrene, S., Siegrist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+bacterial+ribosomes+using+chloramphenicol+and+erythromycin+columns.">Purification of bacterial ribosomes using chloramphenicol and erythromycin columns.</a> Biochimie. 62, 69-77 (1980).</li><li>Meskauskas, A., Petrov, A. N., Dinman, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+functionally+important+amino+acids+of+ribosomal+protein+L3+by+saturation+mutagenesis.">Identification of functionally important amino acids of ribosomal protein L3 by saturation mutagenesis.</a> Mol. Cell Biol. 25, 10863-10874 (2005).</li><li>Algire, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+a+reconstituted+yeast+translation+initiation+system.">Development and characterization of a reconstituted yeast translation initiation system.</a> RNA. 8, 382-397 (2002).</li><li>Maguire, B. A., Wondrack, L. M., Contillo, L. G., Xu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+chromatography+system+to+isolate+active+ribosomes+from+pathogenic+bacteria.">A novel chromatography system to isolate active ribosomes from pathogenic bacteria.</a> RNA. 14, 188-195 (2008).</li><li>Leshin, J. A., Rakauskaite, R., Dinman, J. D., Meskauskas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+purity,+activity+and+structural+integrity+of+yeast+ribosomes+purified+using+a+general+chromatographic+method.">Enhanced purity, activity and structural integrity of yeast ribosomes purified using a general chromatographic method.</a> RNA. Biol. 7, 354-360 (2010).</li><li>Triana, F., Nierhaus, K. H., Chakraburtty, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+RNA+binding+to+80S+ribosomes+from+yeast:+evidence+for+three+sites.">Transfer RNA binding to 80S ribosomes from yeast: evidence for three sites.</a> Biochem. Mol. Biol. Int. 33, 909-915 (1994).</li><li>Azzam, M. E., Algranati, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+puromycin+action:+fate+of+ribosomes+after+release+of+nascent+protein+chains+from+polysomes.">Mechanism of puromycin action: fate of ribosomes after release of nascent protein chains from polysomes.</a> Proc. Natl. Acad. Sci. U. S. A. 70, 3866-3869 (1973).</li><li>Nathans, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Puromuycin+inhibition+of+protein+synthesis:+incorporation+of+puromycin+into+peptide+chains.">Puromuycin inhibition of protein synthesis: incorporation of puromycin into peptide chains.</a> Proc. Natl. Acad. Sci. U. S. A. 51, 585-592 (1964).</li><li>Rabinovitz, M., Fisher, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dissociative+effect+of+puromycin+on+the+pathway+of+protein+synthesis+by+Ehrlich+ascites+tumor+cells.">A dissociative effect of puromycin on the pathway of protein synthesis by Ehrlich ascites tumor cells.</a> J. Biol. Chem. 237, 477-481 (1962).</li><li>Allen, D. W., Zamecnik, P. 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A. 45, 1721-1729 (1959).</li><li>Becker-Ursic, D., Davies, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+and+in+vitro+phosphorylation+of+ribosomal+proteins+by+protein+kinases+from+Saccharomyces+cerevisiae.">In vivo and in vitro phosphorylation of ribosomal proteins by protein kinases from Saccharomyces cerevisiae.</a> Biochemistry. 15, 2289-2296 (1976).</li></ol>

Pas de conflits d&#39;intérêt déclarés.

Protocoles de purification ribosome essentiellement impliquent lyse des cellules, la récolte d&#39;une fraction cytosolique d&#39;une vrille à basse vitesse, puis granulation ribosomes par centrifugation à grande vitesse 1. Bien qu&#39;un peu de nouvelles méthodes ont été utilisées pour la purification de ribosomes bactériens, le même n&#39;avait pas été vrai pour les eucaryotes 2-4. Bien que des mesures supplémentaires ont été ajoutés au fil des années, se lave le sel par exemple, ...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Société </td><td> Numéro de catalogue </td></tr><tr><td> Sulfolink Couplage de résine </td><td> Pierce </td><td> 20402 </td></tr><tr><td> Mini-batteur billes 16 </td><td> Biospec </td><td> 607 </td></tr><tr><td> Optima Max E ultracentrifugeuse </td><td> Beckman Coulter </td><td></td></tr><tr><td> Légende RT </td><td> Sorvall </td><td></td></tr><tr><td> 0,5 mm Perles de verre </td><td> Biospec </td><td> 11079105 </td></tr><tr><td> Tris </td><td> Sigma-Aldrich </td><td> 252859 </td></tr><tr><td> EDTA </td><td> Sigma-Aldrich </td><td> E9884 </td></tr><tr><td> TNT </td><td> Sigma-Aldrich </td><td> 43815 </td></tr><tr><td> HEPES </td><td> Sigma-Aldrich </td><td> 54459 </td></tr><tr><td> NH 4 Cl </td><td> Sigma-Aldrich </td><td> A9434 </td></tr><tr><td> KCl </td><td> Sigma-Aldrich </td><td> 60128 </td></tr><tr><td> Mg (OAc) 2 </td><td> Sigma-Aldrich </td><td> M5661 </td></tr><tr><td> KOH </td><td> Sigma-Aldrich </td><td> 221473 </td></tr><tr><td> Glycérol </td><td> Sigma-Aldrich </td><td> G5516 </td></tr><tr><td> Puromycine </td><td> Sigma-Aldrich </td><td> P7255 </td></tr><tr><td> GTP </td><td> Fermentas </td><td> R0461 </td></tr></tbody></table>

chromatographic purification of highly active yeast ribosomes

Ribosomes eucaryotes sont beaucoup plus labiles par rapport à leurs homologues eubactériennes et archael, posant ainsi un défi important pour les chercheurs. Particulièrement gênant est le fait que la lyse des cellules libère un grand nombre de protéases et nucléases qui peut dégrader les ribosomes. Ainsi, il est important de séparer les ribosomes de ces enzymes aussi rapidement que possible. Malheureusement, les méthodes classiques ultracentrifugation différentielle feuilles ribosomes exposés à ces enzymes pour des périodes trop longues de temps, des répercussions sur leur intégrité structurelle et la fonctionnalité. Pour résoudre ce problème, nous utilisons une méthode chromatographique en utilisant une cystéine chargé Sulfolink résine. Cette application simple et rapide réduit considérablement la co-purifiant activités protéolytiques et nucléolytique, produisant des rendements élevés intacte, les ribosomes levure hautement biochimiquement active. Nous suggérons que cette méthode devrait également être applicable aux ribosomes de mammifères. La simplicité dela méthode, et la pureté améliorée et l&#39;activité des ribosomes purifié par Chromatographie représente un progrès technique important pour l&#39;étude des ribosomes eucaryotes.

Watch this Scientific Journal Video about Chromatographic Purification of Highly Active Yeast Ribosomes at JoVE.com

Chromatographic Purification of Highly Active Yeast Ribosomes

La contamination des préparations de ribosomes eucaryotes purifié par les méthodes traditionnelles par les co-purifiant nucléases et protéases une incidence négative sur l&#39;aval des analyses biochimiques et structurelles. Une méthode de purification chromatographique rapide et simple est utilisée pour résoudre ce problème en utilisant des ribosomes levure comme système modèle.

Purification chromatographique des ribosomes de levure hautement active

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

chromatographic-purification-of-highly-active-yeast-ribosomes

Research

JoVE Journal

Biology

16.7K vues.  University of Maryland. La contamination des préparations de ribosomes eucaryotes purifié par les méthodes traditionnelles par les co-purifiant nucléases et protéases une incidence négative sur l'aval des analyses biochimiques et structurelles. Une méthode de purification chromatographique rapide et simple est utilisée pour résoudre ce problème en utilisant des ribosomes levure comme système modèle.

Vidéo: Chromatographic Purification of Highly Active Yeast Ribosomes

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Coloration des protéines dans les gels de Coomassie G-250, sans solvant et l&#39;acide acétique organique

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Recherche

Biologie

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Purification des mitochondries de cellules de levure

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Preparation of Highly Coupled Rat Heart Mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.

We describe &#1072; protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

Préparation de coeur de rat fortement couplées Mitochondries

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis by antibiotics and during nascent polypeptide synthesis. Some of these antibiotics, and regulatory nascent polypeptides mostly in the form of peptidyl-tRNAs, inhibit either peptide bond formation or translation termination1-7. These inhibitory events can stop the movement of the ribosome, a phenomenon termed "translational arrest". Translation arrest induced by either an antibiotic or a nascent polypeptide has been shown to regulate the expression of genes involved in diverse cellular functions such as cell growth, antibiotic resistance, protein translocation and cell metabolism8-13. Knowledge of how antibiotics and regulatory nascent polypeptides alter ribosome function is essential if we are to understand the complete role of the ribosome in translation, in every organism.
Here, we describe a simple methodology that can be used to purify, exclusively, for analysis, those ribosomes translating a specific mRNA and containing a specific peptidyl-tRNA14. This procedure is based on selective isolation of translating ribosomes bound to a biotin-labeled mRNA. These translational complexes are separated from other ribosomes in the same mixture, using streptavidin paramagnetic beads (SMB) and a magnetic field (MF). Biotin-labeled mRNAs are synthesized by run-off transcription assays using as templates PCR-generated DNA fragments that contain T7 transcriptional promoters. T7 RNA polymerase incorporates biotin-16-UMP from biotin-UTP; under our conditions approximately ten biotin-16-UMP molecules are incorporated in a 600 nt mRNA with a 25% UMP content. These biotin-labeled mRNAs are then isolated, and used in in vitro translation assays performed with release factor 2 (RF2)-depleted cell-free extracts obtained from Escherichia coli strains containing wild type or mutant ribosomes. Ribosomes translating the biotin-labeled mRNA sequences are stalled at the stop codon region, due to the absence of the RF2 protein, which normally accomplishes translation termination. Stalled ribosomes containing the newly synthesized peptidyl-tRNA are isolated and removed from the translation reactions using SMB and an MF. These beads only bind biotin-containing messages.
The isolated, translational complexes, can be used to analyze the structural and functional features of wild type or mutant ribosomal components, or peptidyl-tRNA sequences, as well as determining ribosome interaction with antibiotics or other molecular factors 1,14-16. To examine the function of these isolated ribosome complexes, peptidyl-transferase assays can be performed in the presence of the antibiotic puromycin1. To study structural changes in translational complexes, well established procedures can be used, such as i) crosslinking to specific amino acids14 and/or ii) alkylation protection assays1,14,17.

A major impediment to biochemical analyses of ribosomes containing nascent peptidyl-tRNAs has been the presence of other ribosomes in the same samples, ribosomes not involved in the translation of the specific mRNA sequence being analyzed. We developed a simple methodology to purify, exclusively, the ribosomes containing the nascent peptidyl-tRNA of interest.

Isolement des ribosomes Traduire Contenant Peptidyl-ARNt pour des analyses fonctionnelles et structurelles

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis ...

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

Méthode d&#39;insertion de levure Colony

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

La reconstitution in vitro de l&#39;actif T. La télomérase castaneum

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

Haut débit écran surexpression plasmide de levure

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Écrans concurrentiel des bibliothèques génomiques de levure Barcoded

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Bourgeonnement de levure extraction et purification des protéines pour l&#39;étude de la fonction, Interactions, et des modifications post-traductionnelles

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

Purification non chromatographique de polypeptides recombinants élastine et de leurs fusions avec des peptides et des protéines<em&gt; Escherichia coli</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...