Desideriamo ringraziare tutti i membri del laboratorio Dinman, tra Ashton Belew, Karen Jack, Sharmishtha Musalga, Sergey Sulima, Shivani Reddy, e Michael Rhodin per il loro aiuto e l&#39;ingresso su questo progetto. Questo lavoro è stato sostenuto dalle concessioni a AM dalla American Heart Association (AHA 0630163N) e JDD dal National Institutes of Health (5R01 GM058859-12). JAL è stata sostenuta da un reinvestimento americano e Recovery Act del 2009, supplemento alla concessione genitore (3R01GM058859-11S1).

1. Preparazione di un cisteina carica Sulfolink resina <ol><li> Preparare Sulfolink resina (Pierce) a temperatura ambiente. </li><li> Posizionare un totale di 10 ml di una emulsione al 50% di gel di accoppiamento Sulfolink come fornito dal produttore in due flaconcini da 10 ml in plastica, con 5 ml distribuito in ciascuna provetta. </li><li> Centrifugare brevemente fiale a 850 xg e rimuovere con attenzione surnatanti. </li><li> Lavare il gel tre volte in un tampone di accoppiamento (50 mM Tris, pH 8,5, 5 mM EDTA) da risospendere le perle in 5 ml, centrifugazione a 850 xg brevemente e rimuovere il surnatante tramite pipetta. </li><li> Aggiungere cinque ml di una soluzione 50 mM di L-cisteina in un tampone di accoppiamento a ciascuna provetta e miscelare l&#39;impasto per 1 ora a 25 ° C. </li><li> Rimuovere il residuo di L-cisteina con il lavaggio come descritto al punto 1.4. </li><li> Risospendere il gel in 10 ml di tampone vincolante [20 HEPES mM, pH 7,6, 5 mM Mg (OAC) 2, 60 mM di NH 4 Cl, 2 mM DTT] ed equilibrare la resina con il lavaggio in 10 ml di tampone vincolante3 volte come descritto al punto 1.4. Resina decantare in una centrifuga da 10 ml colonna, calotta e conservati a 4 ° C. La capacità di resina per l&#39;RNA legame è ~ 20 A 260 unità per ml. </li></ol> 2. Purificazione cromatografica dei ribosomi con una cisteina carica Sulfolink resina Nota: Se si lavora con un ceppo dove l&#39;attività della proteasi si rivela essere un problema, cocktail inibizione della proteasi può essere utilizzato in tutti i buffer successive. <ol><li> Mantenere tutti i componenti su ghiaccio e preparare tutte le soluzioni con RNase-free acqua e filtrazione sterile. </li><li> Crescere cellule di lievito durante la notte a metà fase di log (OD 595 = 0,6-1,2) nelle matrici appropriate. Celle a pellet per centrifugazione, e lavare 1 grammo cellule in un tampone di legame e centrifugare a 3700 xg per 5 minuti a 4 ° C. </li><li> Risospendere le cellule in 1 ml di tampone legame con un volume totale approssimativo di 2 ml, e di interrompere con un uguale volume di perle di mm 0,5 di vetro refrigerate a 4 ° C ucantare una Biospec Mini-tallone battitore (Bartlesville, OK). I campioni possono essere suddivisi in tubi multipli, se necessario. </li><li> Rimuovere le cellule ininterrotta, organelli e detriti cellulari mediante centrifugazione a 30.000 xg per 30 minuti in un Beckman-Coulter ultracentrifuga Optima Max E (Fullerton, CA). </li><li> Immediatamente prima dell&#39;uso, pellet la resina per un minuto a 1.000 xg per rimuovere la soluzione di storage. Due ml di resina viene utilizzato per ogni grammo di cellule. </li><li> Rimuovere surnatanti lisato cellulare dallo spin 30.000 xg (S30), avendo cura di minimizzare la contaminazione sia dalla frazione lipidica in cima o detriti cellulari alla base dei tubi, e posto direttamente nel liquame Sulfolink carica. Lo strato lipidico possono essere evitati o per la rimozione o spingendo fuori dalla pipetta dopo che la punta ha attraversato nel surnatante. </li><li> Incubare la miscela S30/Sulfolink sul ghiaccio senza mescolare per 15 minuti. </li><li> Posizionare le colonne in provette da 15 ml e centrifugare a 1000 xg perr un minuto. </li><li> Posizionare le frazioni eluato nuovamente dentro le colonne, mescolare a mano, e incubare per altri 15 minuti in ghiaccio. </li><li> Posizionare le colonne in provette da 15 ml e centrifugare a 1000 xg per un minuto. Eliminare l&#39;eluato. </li><li> Colonne tappo e aggiungere 5 ml di tampone vincolanti. Mescolare le colonne a mano fino risospeso, rimuovere i tappi, e centrifugare la centrifuga colonne a 1.000 xg per un minuto. Ripetere questo protocollo di lavaggio altre due volte. </li><li> Aggiungere 1,5 ml di tampone di eluizione (20 mM HEPES-KOH a pH 7,6, 10 mM Mg (OAC) 2, 500 mM KCl, 2mM DTT, 0,5 mg / ml di eparina) per ogni colonna. Mescolare impasti a mano, e incubare in ghiaccio per 2 minuti </li><li> Colonne posto in nuove provette da 15 ml e centrifugare a 1000 xg per 1 minuto. Raccogliere eluato. Ripetere il passaggio di eluizione una volta di più che il volume finale del campione combinato sono circa 3 ml. Resina utilizzata può essere lavata con tampone di eluizione senza eparina seguita da equilibrio con buffe vincolanter, e conservati in volumi di 10 ml di tampone vincolante a 4 ° C per il riutilizzo in seguito. </li></ol> 3. Puromicina trattamento Nota: Un metodo alternativo per rimuovere contaminanti specie di tRNA è quello di passare da una ricca glucosio glucosio impoverito ai media per promuovere il deflusso ribosoma. Tuttavia, questo non cambia lo stato metabolico delle cellule di lievito, che potrebbero influenzare la funzione dei ribosomi e la concentrazione. Al fine di spogliare i ribosomi da endogene peptidil-tRNA, un trattamento viene eseguito con puromicina. <ol><li> Neutralizzare al pH 7.5 100 mM puromicina soluzione con l&#39;aggiunta di KOH 1M goccia a goccia a temperatura ambiente. </li><li> Aggiungere la soluzione puromicina neutralizzata a concentrazione finale di 1 mM ~ eluato. </li><li> Aggiungere 100 mM GTP ad una concentrazione finale di 1 mm. </li><li> Incubare per 30 minuti a 30 ° C. </li></ol> 4. Purificazione dei ribosomi per sedimentazione attraverso cuscini glicerolo <ol><li> Mettere un mldi tampone cuscino (20 HEPES mM, pH 7,6, 10 mM Mg (OAC) 2, 500 mM KCl, 2 mM DTT, 25% glicerolo) in un tubo 4 volumi ultracentrifuga ml in policarbonato. </li><li> Delicatamente strato puromicina trattati frazioni di eluizione in cima al cuscino, e campioni di centrifugare a 100.000 xg notte a 4 ° C. </li><li> Rimuovere i tubi dal rotore centrifuga, ed aspirare surnatanti. </li><li> Lavare pellet contenenti ribosomi purificati due volte con 1 ml di tampone cuscino. </li><li> Risospendere ribosomi in 100 ml di tampone cuscino dalla rottura dolce utilizzando una bacchetta di vetro. </li><li> Dopo la rottura, coprire i tubi con parafilm e agitare ad una velocità moderata in un vortice stanza fredda per un&#39;ora. </li><li> Trasferire il contenuto in una provetta, centrifugare per 5 minuti a velocità massima a 4 ° C, e rimuovere surnatanti ai tubi fresca. </li><li> Ripetere i passaggi 4,1-4,7 con 2 ml di tampone cuscino e, tranne che 100 microlitri di buffer di memorizzazione (50 mM HEPES-KOH a pH 7,6, 50 mM di NH 4 Cl, 5 mM Mg(OAC) 2, 1 mM DTT, 25% glicerolo) è usato in passi 4.4 e 4.5 invece di buffer di cuscino. </li><li> Quantificare i ribosomi purificati spettrofotometricamente (1 A 260 = 20 pmoli dei ribosomi lievito), e conservare a -80 ° C. I risultati tipici da 1 grammo di lievito sono 300-400 pmoli dei ribosomi. </li></ol> 5. Rappresentante dei risultati: Un esempio di specie di RNA estratto da ciascuna delle tre fasi principali del protocollo è mostrato nella Figura 2. Mentre rRNA sono le specie principali presenti in lisati cellulari totali (T) anche questi contengono un gran numero di altre specie di RNA. Ribosomi purificati dalla colonna Sulfolink (SL) contengono anche una grande quantità di tRNA a causa della elevata affinità del letto della colonna per queste specie. Il trattamento di questa frazione con risultati puromicina in idrolisi di peptidi da peptidil-tRNA, e promuove la dissociazione di queste specie da ribosomi. Il puromicina trattati campioni (Pm) la mancanza di co-purifying specie di tRNA, ribosomi e rappresentano quindi completamente puro. Come riportato in precedenza 8, dette ribosomi sono altamente intatti e biochimicamente attive, che li rende ideali per substrati dettagliate analisi funzionali e strutturali. <img src="/files/ftp_upload/3214/3214fig1.jpg" alt="Figura 1"/> Figura 1. Diagramma di flusso metodo. Purificazione cromatografica dei ribosomi con il cisteina legate sulfolink resina è raffigurato. <img src="/files/ftp_upload/3214/3214fig2.jpg" alt="Figura 2"/> Figura 2. Analisi rappresentante di preparati ribosoma. Totale specie di RNA sono stati estratti da lisati cellulari totali (T), Sulfolink ribosomi purificati (SL) e Sulfolink purificata ribosomi successivamente trattati con puromicina e sedimentate attraverso un cuscino glicerolo (Pm). Corsia M rappresenta segnataglie RNA. Bande che rappresentano rRNA 25S e 18S, così come tRNA e altre specie di RNA sono indicati. Tutticorsie sono stati caricati con 2 mg di RNA.

<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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+puromycin+on+rabbit+reticulocyte+ribosomes.">The effect of puromycin on rabbit reticulocyte ribosomes.</a> Biochim. Biophys. Acta. 55, 865-874 (1962).</li><li>Yarmolinsky, M. B., Haba, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+by+puromycin+of+amino+acid+incorporation+into+protein.">Inhibition by puromycin of amino acid incorporation into protein.</a> Proc. Natl. Acad. Sci. U. S. 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>

Nessun conflitto di interessi dichiarati.

Protocolli di purificazione ribosoma fondamentalmente coinvolgere le cellule di lisi, raccolta una frazione citosolica da un testacoda a bassa velocità, e poi cubettatura ribosomi tramite centrifugazione ad alta velocità 1. Mentre un paio di nuovi metodi sono stati utilizzati per purificare i ribosomi batterici, lo stesso non fosse stato vero per eucarioti 2-4. Anche se ulteriori passi sono stati aggiunti nel corso degli anni, si lava sale ad esempio, e cuscini glicerolo (ad esempio vedi 5),...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Sulfolink Giunto in resina </td><td> Perforare </td><td> 20402 </td></tr><tr><td> Mini bead-beater 16 </td><td> Biospec </td><td> 607 </td></tr><tr><td> Optima Max E ultracentrifuga </td><td> Beckman Coulter </td><td></td></tr><tr><td> Legenda RT </td><td> Sorvall </td><td></td></tr><tr><td> Perle di vetro 0,5 millimetri </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> DTT </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> Glicerina </td><td> Sigma-Aldrich </td><td> G5516 </td></tr><tr><td> Puromicina </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

Ribosomi eucariotici sono molto più labili rispetto alle loro controparti eubatteri e archael, ponendo così una sfida significativa per i ricercatori. Particolarmente fastidioso è il fatto che la lisi delle cellule rilascia un gran numero di proteasi e nucleasi che può degradare i ribosomi. Quindi, è importante separare i ribosomi di questi enzimi nel più breve tempo possibile. Purtroppo, i metodi convenzionali ultracentrifugazione differenziale lascia ribosomi esposti a questi enzimi per periodi troppo lunghi di tempo, che influiscono sulla loro integrità strutturale e la funzionalità. Per risolvere questo problema, utilizziamo un metodo cromatografico utilizzando una cisteina carica Sulfolink resina. Questa applicazione semplice e rapida riduce in modo significativo le attività di co-purificazione proteolitici e nucleolytic, producendo rendimenti elevati di intatto, ribosomi lieviti altamente biochimicamente attivi. Suggeriamo che questo metodo dovrebbe essere applicabile anche ai ribosomi dei mammiferi. La semplicità diil metodo, e la purezza maggiore e l&#39;attività del ribosoma cromatograficamente purificato rappresenta un progresso tecnico significativo per lo studio dei ribosomi eucarioti.

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

Chromatographic Purification of Highly Active Yeast Ribosomes

Contaminazione di preparati di ribosomi eucarioti purificata con metodi tradizionali da parte di co-purificazione nucleasi e proteasi influisce negativamente sulla valle analisi biochimiche e strutturali. Un metodo rapido e semplice purificazione cromatografica viene utilizzato per risolvere questo problema utilizzando ribosomi lievito come sistema modello.

Purificazione cromatografica dei ribosomi di lievito Highly 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

16.8K Views.  University of Maryland. Contaminazione di preparati di ribosomi eucarioti purificata con metodi tradizionali da parte di co-purificazione nucleasi e proteasi influisce negativamente sulla valle analisi biochimiche e strutturali. Un metodo rapido e semplice purificazione cromatografica viene utilizzato per risolvere questo problema utilizzando ribosomi lievito come sistema modello.

Video: 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.

La colorazione delle proteine ​​in gel con Coomassie G-250, senza solventi organici acido acetico e

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

Ricerca

Biologia

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.

Purificazione dei mitocondri da cellule di lievito

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.

Preparazione del Cuore Rat altamente Accoppiato mitocondri

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.

Isolamento dei ribosomi Tradurre contenenti peptidil-tRNA per le analisi funzionali e strutturali

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.

Lievito Colony metodo di inserimento

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.

Ricostituzione in vitro di Active T. castaneum telomerasi

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.

High-throughput iperespressione schermo lievito plasmidi

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.

Schermi competitivo genomica delle Biblioteche di lievito con codice a barre

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.

Germogliamento lievito proteine ​​estrazione e purificazione per lo Studio della funzione, interazioni e modificazioni post-traduzionali

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.

Purificazione non-cromatografico di polipeptidi di elastina ricombinante simili e le loro fusioni con Peptidi e Proteine ​​da<em&gt; Escherichia coli</em

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