Wir möchten alle Mitglieder der Dinman Labor, darunter Ashton Belew, Karen Jack, Sharmishtha Musalga, Sergey Sulima, Shivani Reddy, und Michael Rhodin für ihre Hilfe und Input bei diesem Projekt danken. Diese Arbeit wurde durch Fördermittel unterstützt, von der American Heart Association (AHA 0630163N) AM und von den National Institutes of Health (5R01 GM058859-12) JDD. JAL wurde von einem amerikanischen Reinvestment and Recovery Act of 2009 Ergänzung zu den Eltern zu gewähren (3R01GM058859-11S1) unterstützt.

1. Erstellung eines Cystein berechnet SulfoLink Harz <ol><li> Bereiten SulfoLink Harz (Pierce) bei Raumtemperatur. </li><li> Legen Sie insgesamt 10 ml einer 50% igen Aufschlämmung von SulfoLink Ultraschallgel, wie vom Hersteller in zwei 10 ml Kunststoff-Flaschen geliefert, mit 5 ml in jedes Fläschchen verteilt. </li><li> Kurz Zentrifuge Ampullen bei 850 xg und entfernen Sie vorsichtig Überstände. </li><li> Wash Gel dreimal in Bindungspuffer (50 mM Tris, pH 8,5, 5 mM EDTA) durch Resuspendieren der Beads in 5 ml, kurze Zentrifugation bei 850 xg und Entfernen des Überstandes mit einer Pipette. </li><li> Fügen Sie fünf ml einer 50 mM Lösung von L-Cystein in Kupplungspuffer in jedes Röhrchen und mischen Sie die Suspension 1 Stunde bei 25 ° C. </li><li> Entfernen Sie die verbleibenden L-Cystein durch Waschen wie in Schritt 1.4 beschrieben. </li><li> Resuspendieren das Gel in 10 ml Bindungspuffer [20 mM HEPES, pH 7,6, 5 mM Mg (OAc) 2, 60 mM NH 4 Cl, 2 mM DTT] und äquilibrieren das Harz durch Waschen in 10 ml Bindungspuffer3-mal, wie in Schritt 1.4 beschrieben. Dekantieren Harz in einem 10 ml-Zentrifugenröhrchen Spalte, Kappe und bei 4 ° C. Das Harz Kapazität für RNA-Bindung ~ 20 A 260 Einheiten pro ml. </li></ol> 2. Chromatographische Reinigung von Ribosomen mit einem Cystein berechnet SulfoLink Harz Hinweis: Bei der Arbeit mit einem Stamm, wo Protease-Aktivität erweist sich als ein Problem sein, Proteaseinhibierung Cocktails können in allen nachfolgenden Puffer verwendet werden. <ol><li> Pflegen Sie alle Komponenten auf Eis und bereiten alle Lösungen mit RNase-freiem Wasser-und Sterilfiltration. </li><li> Wachsen Hefezellen über Nacht zum mittleren Log-Phase (OD 595 = 0,6 bis 1,2) in geeigneten Medien. Pellet-Zellen durch Zentrifugation und Waschen 1 Gramm Zellen in Bindungspuffer und Zentrifuge bei 3700 xg für 5 Minuten bei 4 ° C. </li><li> Die Zellen in 1 ml Bindungspuffer, um eine ungefähre Gesamtvolumen von 2 ml, und stören mit dem gleichen Volumen von 0,5 mm Glasperlen gekühlt auf 4 ° C usingen ein Biospec Mini-Bead Beater (Bartlesville, OK). Die Proben können in mehrere Rohre geteilt werden, wie gebraucht. </li><li> Entfernen Sie aufgebrochene Zellen, Organellen und Zelltrümmer durch Zentrifugation bei 30.000 xg für 30 Minuten in einer Beckman-Coulter Optima Max E Ultrazentrifuge (Fullerton, CA). </li><li> Unmittelbar vor dem Gebrauch Pellet der Harz für 1 Minute bei 1.000 xg, um die Storage-Lösung zu entfernen. Zwei ml Harz ist für jeden ein Gramm Zellen verwendet. </li><li> Entfernen Zelllysat Überstände von den 30.000 xg Spin (S30), wobei darauf zu Verunreinigungen entweder aus der Lipid-Fraktion an die Spitze oder die Zelltrümmer am Boden der Rohre, und direkt in die Rechnung SulfoLink Gülle zu minimieren. Die Lipidschicht kann entweder durch Beseitigung oder durch Ausstoßen aus der Pipette nach der Spitze in den Überstand überquert hat vermieden werden. </li><li> Inkubieren Sie die S30/Sulfolink Mischung auf Eis ohne Mischen für 15 Minuten. </li><li> Legen Sie die Spalten in 15 ml konischen Röhrchen und zentrifugieren bei 1.000 xg fürr 1 Minute. </li><li> Setzen Sie den Durchfluss Fraktionen zurück in die Spalten, mischen von Hand, und inkubieren für weitere 15 Minuten auf Eis. </li><li> Legen Sie die Spalten in 15 ml konischen Röhrchen und zentrifugieren bei 1.000 xg für 1 Minute. Entsorgen Sie den Durchfluss. </li><li> Cap Spalten und 5 ml Bindungspuffer. Mix Spalten von Hand bis resuspendiert, entfernen Sie den Deckel und zentrifugieren Sie die Spalten Zentrifuge bei 1.000 xg für 1 Minute. Wiederholen Sie diesen Wasch-Protokoll zwei weitere Male. </li><li> Fügen Sie 1,5 ml Elutionspuffer (20 mM HEPES-KOH, pH 7,6, 10 mM Mg (OAc) 2, 500 mM KCl, 2 mM DTT, 0,5 mg / ml Heparin), um jede Spalte. Mix Schlämme von Hand, und auf Eis inkubieren für 2 Minuten </li><li> Legen Sie Spalten in neue 15 ml konischen Röhrchen und zentrifugieren bei 1.000 xg für 1 Minute. Sammeln Eluat. Wiederholen Sie den Elutions noch einmal so, dass die endgültige Kombination Probenvolumina ~ 3 ml sind. Gebrauchte Harz kann mit Elutionspuffer ohne Heparin gewaschen und anschließend durch Äquilibrierung mit verbindlichen buffer, und in 10 ml Volumen Bindungspuffer bei 4 ° C für die Wiederverwendung später gespeichert. </li></ol> 3. Puromycin-Behandlung Hinweis: Eine alternative Methode zur Entfernung kontaminierender tRNA-Spezies ist es, aus einer Glucose reich an Glucose verbraucht Medien Ribosom Abfluss zu fördern wechseln. Allerdings ändert das die metabolischen Status der Hefezellen, die Ribosomen-Funktion und Konzentration beeinträchtigen könnten. Um die Ribosomen aus endogenen Peptidyl-tRNA, eine Behandlung mit Puromycin durchgeführt Streifen. <ol><li> Neutralisieren auf pH 7,5 100 mM Puromycin Lösung durch Zugabe von 1M KOH tropfenweise bei Raumtemperatur. </li><li> Add neutralisiert Puromycin Lösung ~ 1mm Endkonzentration im Eluat. </li><li> Add 100 mM GTP zu einer Endkonzentration von 1 mM. </li><li> Inkubieren für 30 Minuten bei 30 ° C. </li></ol> 4. Reinigung von Ribosomen durch Sedimentation durch Glycerin Kissen <ol><li> Legen Sie eine mlvon Polster-Puffer (20 mM HEPES, pH 7,6, 10 mM Mg (OAc) 2, 500 mM KCl, 2 mM DTT, 25% Glycerin) in ein 4 ml Volumen Polycarbonat Ultrazentrifuge Röhre. </li><li> Vorsichtig Schicht Puromycin Elutionsfraktionen auf der Oberseite des Kissens und Zentrifuge Proben bei 100.000 xg bei 4 ° C über Nacht behandelt </li><li> Nehmen Sie Rohre aus dem Zentrifugenrotor, und saugen Überstände. </li><li> Wash Pellets gereinigt Ribosomen zweimal mit 1 ml der Kissen-Puffer. </li><li> Resuspendieren Ribosomen in 100 ul Kissen Puffer durch vorsichtiges Störung mit einem Glasstab. </li><li> Nach Unterbrechungen, decken Rohre mit Parafilm und schütteln mit mäßiger Geschwindigkeit in einem kalten Raum Wirbel für eine Stunde. </li><li> Übertragen Sie den Inhalt in ein Mikrozentrifugenröhrchen, Zentrifuge für 5 Minuten bei maximaler Geschwindigkeit bei 4 ° C, und entfernen Sie Überstände an die frische Röhren. </li><li> Wiederholen Sie die Schritte von 4,1 bis 4,7 mit 2 ml Puffer und Polster, außer dass 100 ul der Pufferspeicher (50 mM HEPES-KOH pH 7,6, 50 mM NH 4 Cl, 5 mM Mg(OAc) 2, 1 mM DTT, 25% Glycerin) in Schritten 4.4 und 4.5 statt Kissen Puffer verwendet. </li><li> Quantifizierung der gereinigten Ribosomen spektrophotometrisch (1 A 260 = 20 pMol Hefe Ribosomen) und bei -80 ° C. Typische Ergebnisse von 1 Gramm Hefe sind 300-400 pmol Ribosomen. </li></ol> 5. Repräsentative Ergebnisse: Ein Beispiel für RNA-Spezies aus jedem der drei großen Schritte des Protokolls extrahiert wird in Abbildung 2 dargestellt. Während rRNAs sind die wichtigsten Arten, die in insgesamt Zelllysaten (T) enthält auch diese eine große Anzahl von anderen RNA-Spezies. Ribosomen aus dem SulfoLink Spalte (SL) gereinigt enthalten auch eine große Menge von tRNAs durch die hohe Affinität der Spalte Bett für diese Arten auch. Die Behandlung dieser Fraktion mit Puromycin führt zur Hydrolyse von Peptiden aus Peptidyl-tRNAs und fördert Dissoziation dieser Arten aus Ribosomen. Die Puromycin behandelten Proben (Pm) mangelnde Zusammenarbeit purifying tRNA-Spezies, und stellen damit vollkommen rein Ribosomen. Wie bereits 8 berichtet, sind diese Ribosomen sehr intakt und biochemisch aktiver, wodurch sie ideal Substrate für detaillierte funktionelle und strukturelle Analysen. <img src="/files/ftp_upload/3214/3214fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Methode Flowchart. Chromatographische Reinigung von Ribosomen mit dem Cystein verknüpft SulfoLink Harz dargestellt. <img src="/files/ftp_upload/3214/3214fig2.jpg" alt="Abbildung 2"/> Abbildung 2. Vertreter Analyse von Ribosomen Zubereitungen. Gesamt-RNA Spezies wurden aus insgesamt Zelllysaten (T), SulfoLink gereinigt Ribosomen (SL) und SulfoLink gereinigt Ribosomen anschließend mit Puromycin behandelt extrahiert und sedimentiert durch eine Glycerin Kissen (PM). Lane M steht für RNA-Größenmarker. Bands vertreten 25S-und 18S-rRNA, sowie tRNAs und anderen RNA-Spezies sind angegeben. AlleBahnen wurden mit 2 pg RNA geladen.

<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>

Keine Interessenskonflikte erklärt.

Ribosomen Aufreinigungsprotokolle grundsätzlich um Zellen zu lysieren, die Ernte einer cytosolischen Fraktion aus einer niedrigen Geschwindigkeit drehen, und dann Pelletierung Ribosomen durch Hochgeschwindigkeitszentrifugation 1. Während ein paar neue Methoden zur Reinigung von bakteriellen Ribosomen verwendet wurden, hatte das gleiche nicht für Eukaryoten 2-4 wahr. Obwohl zusätzliche Schritte im Laufe der Jahre, wie zB Salz wäscht, und Glycerin Kissen (siehe zB 5) hinzugefügt word...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> SulfoLink Coupling Harz </td><td> Durchstechen </td><td> 20402 </td></tr><tr><td> Mini Bead-Beater 16 </td><td> Biospec </td><td> 607 </td></tr><tr><td> Optima Max E Ultrazentrifuge </td><td> Beckman Coulter </td><td></td></tr><tr><td> Legend RT </td><td> Sorvall </td><td></td></tr><tr><td> 0,5 mm Glasperlen </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> Glycerol </td><td> Sigma-Aldrich </td><td> G5516 </td></tr><tr><td> Puromycin </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

Eukaryotischen Ribosomen sind sehr viel labiler als ihre eubakterielle und archael Pendants verglichen, so dass damit eine bedeutende Herausforderung für die Forscher. Besonders störend ist die Tatsache, dass die Lyse von Zellen eine große Anzahl von Proteasen und Nukleasen, die Ribosomen abbauen können Releases. Daher ist es wichtig, dass Ribosomen aus dieser Enzyme so schnell wie möglich zu trennen. Leider lässt herkömmlicher Differenzialsperren Ultrazentrifugation Methoden Ribosomen, die mit diesen Enzymen für unvertretbar lange Zeiträume, die Einfluss auf die strukturelle Integrität und Funktionalität. Um dieses Problem anzugehen, setzen wir ein chromatographisches Verfahren mit einem Cystein berechnet SulfoLink Harz. Diese einfache und schnelle Anwendung deutlich reduziert Co-Reinigung proteolytische und nucleolytischen Aktivitäten, hohe Erträge von intakten, sehr biochemisch aktive Hefe Ribosomen. Wir schlagen vor, dass diese Methode auch anwendbar sein Säugetier-Ribosomen. Die Einfachheit derdie Methode und die verstärkte Reinheit und Aktivität der chromatographisch gereinigten Ribosomen stellt einen bedeutenden technischen Fortschritt für das Studium der eukaryotischen Ribosomen.

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

Chromatographic Purification of Highly Active Yeast Ribosomes

Die Kontamination von Zubereitungen der eukaryotischen Ribosomen mit herkömmlichen Methoden durch Co-Reinigung Nukleasen und Proteasen gereinigt negativ auf die nachgelagerten biochemische und strukturelle Analysen. Eine schnelle und einfache chromatographische Reinigungsverfahren wird verwendet, um dieses Problem unter Verwendung von Hefe Ribosomen als Modellsystem zu lösen.

Chromatographische Reinigung von hoch aktiven Hefe Ribosomen

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 Aufrufe.  University of Maryland. Die Kontamination von Zubereitungen der eukaryotischen Ribosomen mit herkömmlichen Methoden durch Co-Reinigung Nukleasen und Proteasen gereinigt negativ auf die nachgelagerten biochemische und strukturelle Analysen. Eine schnelle und einfache chromatographische Reinigungsverfahren wird verwendet, um dieses Problem unter Verwendung von Hefe Ribosomen als Modellsystem zu lösen.

Serie: 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.

Färbung von Proteinen in Gelen mit Coomassie G-250 ohne organische Lösungsmittel und Essigsäure

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

Forschung

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.

Reinigung von Mitochondrien aus Hefezellen

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.

Herstellung von hoch Coupled Rat-Mitochondrien

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.

Isolierung von Ribosomen übersetzen enthaltend Peptidyl-tRNAs für funktionelle und strukturelle Analysen

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.

Hefekolonie Embedding-Methode

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.

In vitro Rekonstitution der 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 ...

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 Hefeplasmid Überexpression Bildschirm

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.

Competitive Genomic Screens von Barcoded Hefe Bibliotheken

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.

Bäckerhefe Protein Extraktion und Reinigung für das Studium der Funktion, Wechselwirkungen, und post-translationale Modifikationen

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.

Nicht-chromatographischen Reinigung von rekombinantem Elastin-ähnliche Polypeptide und deren Fusionen mit Peptiden und Proteinen aus<em&gt; Escherichia coli</em

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