Wir danken für die technische Unterstützung von Sini Miettinen, Minna Pöllänen und Taru Rautavesi. Wir bedanken uns für die Bereitstellung Helka Nurkkala HEK 293-Zellen, die Twin-Strep-getaggte GFP und Pekka Evijärvi für die Bereitstellung von Aufnahmegerät. Diese Arbeit wurde von der Akademie von Finnland finanziert, gewähren Nummern 138329, 134684 und 258978.

1. Vernetzung von Strep-Tactin Polymethacrylatharz mit Bis (sulfosuccinimidyl) suberat (BS3) <ol><li> Äquilibrieren einer abgedichteten Mikroröhrchen, das 2 mg BS3 Vernetzer auf Raumtemperatur. ACHTUNG: BS3 ist ein gefährlicher Stoff. Schutzhandschuhe und Schutzbrille tragen. </li><li> Resuspendieren Strep-Tactin Polymethacrylatharz (50% Suspension in 100 mM Tris-HCl, pH 8,0; 1 mM EDTA; 150 mM NaCl) durch kurzes kräftiges Schütteln und sofort übertragen 600 ul der Suspension auf eine Spin-Säule mit einer Pipettenspitze mit der Ende abgeschnitten. </li><li> Zentrifugieren bei 1500 × g für 30 Sekunden bei Raumtemperatur. Verwerfen Sie den Durchfluss und mit 450 ul phosphatgepufferter Salzlösung (PBS) auf die Säule. </li><li> Wiederholen Sie den vorherigen Schritt 2 mehrmals, um Tris-Puffer vollständig ersetzen mit PBS und lassen Sie den Harz in 430 ul PBS auf pH 8,0 nach der letzten Zentrifugation angepasst. </li><li> Punktieren Sie die Folie des Mikroröhrchens mit BS3 mit einer Pipettenspitze, die 1001, l Reinstwasser. Lösen Sie das Pulver in Wasser BS3 durch vorsichtiges Auf-und Abpipettieren und sofort 20 ul der Lösung für das Spin-Säule. Die Endkonzentration von BS3 in der Vernetzungsreaktion ist ~ 1,2 mm. </li><li> Drehen der Säule für 30 Minuten bei Raumtemperatur. Überprüfen Sie, dass das Harz richtig mit dem BS3-Lösung gemischt. </li><li> Um die Reaktion abzuschrecken, addieren 6 ul 3 M Tris-HCl, pH 7,5, und dreht die Säule weitere 15 min bei Raumtemperatur. </li><li> Zentrifugieren bei 1500 × g für 30 Sekunden bei Raumtemperatur. Verwerfen Sie den Durchfluss und Resuspendieren des vernetzten Harzes in 450 ul Tris-gepufferter Kochsalzlösung mit Tween 20 (TBST). Wiederholen Sie die Zentrifugation und Waschschritte zwei weitere Male. Im letzten Schritt, resuspendieren Harz in 450 ul TBS. </li><li> Übertragen Sie die Harzsuspension in ein frisches Röhrchen mit einer Pipettenspitze mit dem Ende abgeschnitten. Resuspendieren des Harzes in der Säule mit weiteren 450 &amp;mgr; l TBS links und übertragen auf den gleichen Rohr. Wiederholen the letzten Schritt nochmals eine maximale Übertragung des Harzes aus der Säule, um das Rohr zu gewährleisten. </li><li> Lassen Sie den Schlauch stehen für 10 Minuten bei Raumtemperatur und die Lautstärke auf 600 ul indem überschüssiges TBS. Das Harz ist bereit für den sofortigen Einsatz (empfohlen) oder können bei 4 ° C gelagert werden, ohne zu frieren. </li></ol> 2. Bindung des Twin-Strep-markierten Protein-Köder und assoziierten Komplexe auf die Cross-linked Strep-Tactin Polymethacrylatharz <ol><li> Zentrifuge 1 ml der Probe Köderprotein in Bindungspuffer bei 17.000 × g für 10 min bei 4 ° C und den Überstand in ein frisches Röhrchen. </li><li> Zur Minimierung der Bindung von endogenem biotinylierten Proteine ​​an die Strep-Tactin Harz, Zugabe von Avidin zu einer Endkonzentration von 100 ug / ml und dreht 15 min bei 4 ° C. <ol><li> Wenn das vernetzte Harz aus Schritt 1,10 für einen längeren Zeitraum bei 4 ° C gelagert wurden, sammeln das Harz durch Zentrifugation bei 400 xg for 1 min bei 4 ° C, Überstand verwerfen und das Harz mit 1 ml TBST. Wiederholen der Zentrifugations-und Waschschritte noch zweimal, zuerst mit TBST und dann mit TBS gewaschen. Das Röhrchen bei 400 × g für 1 min bei 4 ° C und Einstellen auf das ursprüngliche Volumen, indem überschüssiges TBS. </li></ol></li><li> Resuspendieren des vernetzten Strep-Tactin Harz durch Vortexen. Sofort 50 ul der Harzsuspension dem Rohr den Köder-Protein enthaltenden Probe mit einem geschnittenen Pipettenspitze und drehen noch 30 min bei 4 ° C. </li><li> Während der Wartezeit eingestellt Thermomixer auf 55 ° C vorheizen und 500 ul Elutionspuffer zur Verwendung in Schritt 3.1. </li><li> Zentrifuge bei 400 × g für 1 min bei 4 ° C. Verwerfen des Überstandes und Waschen des Harzes auf einem Rotator für 5 min bei 4 ° C mit 1 ml vorgekühltes Waschpuffer # 1. Wiederholen Sie die Zentrifugation und Waschschritte dreimal. Im letzten Schritt, resuspendieren Harz in 250 ul Waschpuffer # 2. </li><li> Tr<html> ansfer die Harzsuspension in einen frischen Spin-Säule. Resuspendieren des restlichen in dem Rohr in einem anderen 250 ul Waschpuffer # 2 Harz und übertragen auf den gleichen Spalte. </li><li> Zentrifuge bei 400 g für 3 min bei 4 ° C, entsorgen Sie die Durchfluss und übertragen Sie die Spalte zu einem frischen Delphin-Nase-2-ml-Tube. Gehen Sie sofort auf die Elution Schritt unten. </li></ol> 3. Spezifische Elution der Proteinkomplexe <ol><li> Jeweils 150 ul der vorgewärmten Elutionspuffer von Schritt 2.4, um die Spin-Säule. </li><li> Inkubation in Thermomixer für 5 min bei 55 ° C, bei 1.400 Umdrehungen pro Minute zittern. </li><li> Zentrifugieren bei 1500 × g für 1 min bei Raumtemperatur. </li><li> Entsorgen Sie die Spalte und speichern die gereinigten Zielproteine ​​bei ≤ -20 ° C </li></ol><table border="0" cellpadding="0" cellspacing="0" width="598"><tbody><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> Phosphat-gepufferte Salzlösung (PBS) </td></tr>0 &quot;&gt; <td height="20" style="height:20px;width:299px;"> Na 2 HPO 4 </td><td style="width:299px;"> 10 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> KH 2 PO 4 </td><td style="width:299px;"> 2 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> NaCl </td><td style="width:299px;"> 137 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> KCl </td><td style="width:299px;"> 2,7 mM </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> pH-Wert auf 7,4 eingestellt, sofern nicht anders angegeben (pH 8.0 in Abschnitt 1.4) </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> Tris-gepufferter Salzlösung (TBS) </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Tris-HCl, pH 7,4 </td><td style="width:299px;"> 50 mM </td></tr><tr height="20"><td hei<html> ght = &quot;20&quot; style = &quot;height: 20px; width: 299px;&quot;&gt; NaCl </td><td style="width:299px;"> 150 mM </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> Tris-gepufferte Kochsalzlösung mit Tween 20 (TBST) </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Tris-HCl, pH 7,4 </td><td style="width:299px;"> 50 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> NaCl </td><td style="width:299px;"> 150 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Tween 20 </td><td style="width:299px;"> 0,1% (v / v) </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> Bindungspuffer </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Tris-HCl, pH 8,0 </td><td style="width:299px;"> 25 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> NaCl </td>x; &quot;&gt; 550 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> NaF </td><td style="width:299px;"> 5 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> EDTA </td><td style="width:299px;"> 0,5 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Glycerol </td><td style="width:299px;"> 10% (v / v) </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> PMSF </td><td style="width:299px;"> 0,1 mM </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> Waschpuffer # 1 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Tris-HCl, pH 8,0 </td><td style="width:299px;"> 25 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> NaCl </td><td style="width:299px;"> 500 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> NaF </<html> td&gt; <td style="width:299px;"> 5 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> EDTA </td><td style="width:299px;"> 0,4 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Igepal CA-630 </td><td style="width:299px;"> 0,2% (v / v) </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Glycerol </td><td style="width:299px;"> 5% (v / v) </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> PMSF </td><td style="width:299px;"> 0,1 mM </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> Waschpuffer # 2 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Tris-HCl, pH 8,0 </td><td style="width:299px;"> 25 mM </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> NaCl </td><td style="width:299px;"> 150 mM </td></tr><tr height="20">2 &quot;height =&quot; 20 &quot;style =&quot; height: 20px; width: 597px; &quot;&gt; Elutionspuffer </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> Tris-HCl, pH 8,0 </td><td style="width:299px;"> 25 mM </td></tr><tr height="22"><td height="22" style="height:22px;width:299px;"> SDS </td><td style="width:299px;"> 1% (w / v) </td></tr></tbody></table> Tabelle 1. Puffer in der vorliegenden Studie verwendet.

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Das obige Protokoll kann verwendet werden, um jede Doppel Strep-getaggte Köder Protein von Interesse und die damit verbundenen Komplexe in jeder geeigneten Puffer, die nicht Biotin oder starke Vergällungsmittel enthält reinigen. In der aktuellen Version des Protokolls, Bindung und Waschen unter relativ stringenten Bedingungen in der Gegenwart von hohen Salz und nicht-ionischen Detergens durchgeführt. Dies führt zwar zu weniger Hintergrund, kann fragilen Proteinkomplexe unter diesen Bedingungen zu distanzieren. Um s...

In den letzten Jahren hat Strep-Tag-Technologie in vielen Bereichen der biomedizinischen Forschung, einschließlich der Proteomforschung und Strukturbiologie weit verbreitet. Diese Technologie zur Proteinreinigung, die auf der Fusion von rekombinanten Proteinen an einer kurzen Strep-tag-Peptid beruht, mit dem Aufkommen von Affinitätsmatrizes Durchführung Strep-Tactin, eine gentechnisch Variante von Streptavidin mit verbesserter Peptid-Bindungsfähigkeit 1, 2 gereift. Fusionsproteine, zwei Kopien von Strep-tag II, Twin-Strep-Tag oder SIII-Tag, zeigen eine höhere Affinität für Strep-Tactin Matrizen als solche, die nur eine einzige Strep-Tag, eine effizientere Reinigung der rekombinanten Proteine ​​und bezeichnet enthält ihre zugeordneten Bindungspartnern. Allerdings hat der höhere Affinität von Twin-Strep-markierte Proteine ​​zu Strep-Tactin auch seine Kehrseite. Kompetitive Elution derartiger Proteine ​​mit einem Überschuss Biotin kann unvollständig sein, was zu einer verringerten Ausbeute Zielprotein. A mErz effiziente Alternative ist die Elution mit SDS, aber es führt zu unerwünschten Kontamination der Probe mit Strep-Tactin aus dem Harz freigesetzt, so dass der Test nicht mit Proteomanalyse. Dieser Artikel stellt eine Technik, um diese Beschränkung durch die ersten Stabilisierungs harzgekuppelten Tetramer von Strep-Tactin durch chemische Vernetzung und dann unter Verwendung von SDS zur Doppel-Strep-markierte Proteine ​​und ihre zugehörigen Komplexe aus der resultierenden vernetzten Harz zu eluieren überwinden. Somit kann eine ausreichende Proteinausbeute ohne Kontamination der Probe mit Strep-Tactin erreicht werden, wodurch eine weitere Analyse durch Massenspektrometrie erlaubt. Das Verfahren ist mit einer oberflächenexponierten SIII-Tag 3 oder Twin-Strep-Tag (Aminosäuresequenz WSHPQFEK (GGGS) 3 WSHPQFEK und SAWSHPQFEK (GGGS) 2 GGSAWSHPQFEK jeweils) geeignet für die Reinigung von jedem rekombinantes Fusionsprotein. Das Protein kann aus tierischen, pflanzlichen oder bakteriellen Ursprungs sein und kann entweder aus insgesamt Zelle isoliert werdenLysat oder angereicherten Organell-Fraktion. Als Beispiel beschreiben wir hier die Reinigung eines SIII-markierten Protein VPg-Pro von Kartoffelvirus A (PVA) 4 von der Kernfraktion von PVA-infizierten Nicotiana benthamiana Pflanzen. Die Kernfraktion wurde isoliert, wie zuvor beschrieben 5, mit den folgenden Modifikationen: die Zellen wurden nicht mit Formaldehyd behandelt wurde Natriumbutyrat in alle Puffer mit 5 mM Natriumfluorid ersetzt wurde komplette Proteaseinhibitor PMSF substituiert mit Triton X-100-Konzentration im Extraktions Puffer # 2 wurde auf 0,3% gesenkt (v / v) und das Kernpellet durch Zentrifugation über Saccharose-Kissen (Extraktionspuffer # 3) erhalten wurde in 1,45 ml vorgekühltem Bindungspuffer resuspendiert und gedreht wird 1,5 Stunden bei 4 ° C. Die daraus resultierende Kernextrakt die SIII-markierten Köderprotein und die damit verbundenen Komplexe (Köder Proteinprobe) enthält, wurde gemäß dem unten beschriebenen (siehe Abschnitt 2)-Protokoll verarbeitet.

<table border="0" cellpadding="0" cellspacing="0" width="606">
	<tbody>
		<tr>
			<td>Bis(sulfosuccinimidyl) suberate (BS3), No-Weigh format, 8 x 2mg</td>
			<td>Pierce/Thermo Scientific</td>
			<td>21585</td>
			<td><a href="http://www.fishersci.com">www.fishersci.com CAUTION: Hazardous substance. Causes serious respiratory, skin and eye irritation. Wear protective gloves and eye protection.</a></td>
		</tr>
		<tr>
			<td>Strep-Tactin MacroPrep resin (50% suspension)</td>
			<td>IBA</td>
			<td>2-1505</td>
			<td><a href="http://www.iba-lifesciences.com/">www.iba-lifesciences.com</a></td>
		</tr>
		<tr>
			<td>Spin-X centrifuge tube filter, cellulose acetate membrane, pore size 0.45 &#956;m, non-sterile</td>
			<td>Costar (Corning)</td>
			<td>8163</td>
			<td><a href="http://www.corning.com/lifesciences/">www.corning.com/lifesciences/</a></td>
		</tr>
		<tr>
			<td>Dolphin-nose tubes</td>
			<td>Costar (Corning)</td>
			<td>3213</td>
			<td><a href="http://www.corning.com/lifesciences/">www.corning.com/lifesciences/</a></td>
		</tr>
		<tr>
			<td>Avidin&#160;</td>
			<td>IBA</td>
			<td>2-0204</td>
			<td><a href="http://www.iba-lifesciences.com/">www.iba-lifesciences.com</a></td>
		</tr>
	</tbody>
</table>

one-step purification of twin-strep-tagged proteins and their complexes on strep-tactin resin cross-linked with bis(sulfosuccinimidyl) suberate (bs3)

Affinitätsreinigung von Strep-markierten Fusionsproteinen auf Harze Durchführung eines manipulierten Streptavidin (Strep-Tactin) hat sich eine weit verbreitete Methode zur Isolierung von Proteinkomplexen unter physiologischen Bedingungen. Fusionsproteine, zwei Kopien von Strep-tag II, Twin-Strep-Tag oder SIII-Tag bezeichnet, enthalten, haben den Vorteil der höheren Affinität für Strep-Tactin Vergleich zu denen nur eine einzige Strep-Tag enthält, so dass eine effizientere Proteinreinigung. Allerdings wird dieser Vorteil durch die Tatsache, dass die Elution von Zwillings-Strep-markierte Proteine ​​mit Biotin kann unvollständig sein, was zu einer geringen Proteinrückgewinnung auszugleichen. Die Rückgewinnung kann durch die Verwendung stark denaturierender Elution mit Natriumdodecylsulfat (SDS) verbessert werden, aber dies führt zu einer Kontamination mit Strep-Tactin aus dem Harz freigesetzt abzutasten, so dass der Test nicht mit nachgeschalteten Proteomanalyse. Um diese Einschränkung zu überwinden, haben wir ein Verfahren, bei dem mit Harz gekoppelt Tetramer von Strep-Tactin entwickeltenZunächst wird durch kovalente Quervernetzung mit Bis (sulfosuccinimidyl) suberat (BS3) und die resultierende vernetzte Harz wird dann verwendet, um Zielprotein-Komplexe in einer einzigen Charge zu reinigen Reinigungsschritt stabilisiert. Effiziente Elution mit SDS-Protein sorgt für eine gute Erholung, während der Abwesenheit von kontaminierenden Strep-Tactin ermöglicht Downstream-Protein-Analyse durch Massenspektrometrie. Als Proof of Concept, beschreiben wir hier ein Protokoll für die Reinigung von SIII-markierten viralen Protein VPg-Pro aus Kernen von Virus-infizierten N. benthamiana-Pflanzen mit dem Strep-Tactin Polymethacrylatharz mit BS3 vernetzt. Das gleiche Protokoll kann verwendet werden, um jede Doppel Strep-markierten Protein von Interesse reinigen und zu charakterisieren seine physiologischen Bindungspartner werden.

Das Reinigungsverfahren ist schematisch in Fig. 1 dargestellt, zusammen mit einer Darstellung von Problemen mit bestehenden Reinigungsverfahren verbunden sind. <img alt="Figur 1" fo:content-width="5.5in" src="/files/ftp_upload/51536/51536fig1highres.jpg" width="650" /> 1. Schematische Darstellung des Reinigungsverfahrens. Der Workflow umfasst die Stabilisierung des harzgekuppelten Strep-Tactin Tetramer über kovalen...

Watch this Scientific Journal Video about One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3 at JoVE.com

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3

Es wird ein Verfahren zur effizienten Reinigung von Doppel Strep-tag-Fusionsproteine ​​und deren Komplexe spezifisch auf modifizierte Streptavidin beschrieben (Strep-Tactin)-Harz kovalent mit Bis (sulfosuccinimidyl) suberat (BS3) vernetzt. Das Verfahren hat die Vorteile der schnellen Geschwindigkeit, gute Zielprotein Recovery und hohe Reinheit und ist mit nachfolgender Analyse durch Massenspektrometrie kompatibel.

Ein-Schritt-Reinigung von Twin-Strep-markierten Proteine ​​und ihre Komplexe auf Mit Bis (Sulfosuccinimidyl) Strep-Tactin Resin Vernetzte suberat (BS3)

Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for ...

one-step-purification-twin-strep-tagged-proteins-their-complexes-on

Research

JoVE Journal

Biology

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bis(sulfosuccinimidyl) Suberate (BS3)

18.6K Aufrufe.  University of Helsinki.  Es wird ein Verfahren zur effizienten Reinigung von Doppel Strep-tag-Fusionsproteine ​​und deren Komplexe spezifisch auf modifizierte Streptavidin beschrieben (Strep-Tactin)-Harz kovalent mit Bis (sulfosuccinimidyl) suberat (BS3) vernetzt. Das Verfahren hat die Vorteile der schnellen Geschwindigkeit, gute Zielprotein Recovery und hohe Reinheit und ist mit nachfolgender Analyse durch Massenspektrometrie kompatibel. 

Serie: One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)1, and the negative staining has been performed by Wu et al. (2007).2

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.

Reinigung und Visualisierung von Influenza A Virus Ribonukleoproteinkomplexe

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

Forschung

Biologie

Identification of protein complexes with quantitative proteomics in S. cerevisiae

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

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

Identifizierung von Protein-Komplexen mit der quantitativen Proteomik in S. cerevisiae

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

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 ...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Genetic Studies of Human DNA-Reparatur-Proteinen unter Verwendung von Hefe als Modellsystem

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

One-step Metabolomics: Carbohydrates, Organic and Amino Acids Quantified in a Single Procedure

Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on tandem mass spectrometry and quantifies acylcarnitines as a screen for organic acidemias and also measures amino acids. All states also perform enzymatic testing for carbohydrate disorders such as galactosemia. Because the results can be non-specific, follow-up testing of positive results is required using a more definitive method. The present report describes the "urease" method of sample preparation for inborn error screening. Crystalline urease enzyme is used to remove urea from body fluids which permits most other water-soluble metabolites to be dehydrated and derivatized for gas chromatography in a single procedure. Dehydration by evaporation in a nitrogen stream is facilitated by adding acetonitrile and methylene chloride. Then, trimethylsilylation takes place in the presence of a unique catalyst, triethylammonium trifluoroacetate. Automated injection and chromatography is followed by macro-driven custom quantification of 192 metabolites and semi-quantification of every major component using specialized libraries of mass spectra of TMS derivatized biological compounds. The analysis may be performed on the widely-used Chemstation platform using the macros and libraries available from the author. In our laboratory, over 16,000 patient samples have been analyzed using the method with a diagnostic yield of about 17%--that is, 17% of the samples results reveal findings that should be acted upon by the ordering physician. Included in these are over 180 confirmed inborn errors, of which about 38% could not have been diagnosed using previous methods.

The urease method of sample preparation for GC/MS analysis of intermediary metabolites is presented by its inventor. The method allows one-step follow-up of newborn screening for inborn errors by tandem mass spectrometry by quantifying carbohydrates, organic and amino acids all in a single process.

Ein-Schritt-Metabolomics: Kohlenhydrate, organische und Aminosäuren in einem einzigen Verfahren Quantifizierte

Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on ...

Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for magnetic resonance imaging (MRI). Many commercially available ligands are especially useful because they contain 
functional groups that allow for fast, high-purity, and high-yielding conjugation to macromolecules and biomolecules via amine-reactive 
activated esters and isothiocyanate groups or thiol-reactive maleimides. While metalation of these ligands is considered common 
knowledge in the field of bioconjugation chemistry, subtle differences in metalation procedures must be taken into account when 
selecting metal starting materials. Furthermore, multiple options for purification and characterization exist, and selection of the most 
effective procedure partially depends on the selection of starting materials. These subtle differences are often neglected in published 
protocols. Here, our goal is to demonstrate common methods for metalation, purification, and characterization of lanthanide complexes 
that can be used as contrast agents for MRI (Figure 1). We expect that this publication will enable biomedical scientists to incorporate 
lanthanide complexation reactions into their repertoire of commonly used reactions by easing the selection of starting materials and 
purification methods.

We demonstrate the metalation, purification, and characterization of lanthanide complexes. The complexes described here can be conjugated to macromolecules to enable tracking of these molecules using magnetic resonance imaging.

Herstellung, Reinigung und Charakterisierung von Lanthanoidkomplexe für die Verwendung als Kontrastmittel für die Magnetresonanz-Tomographie

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Identifizierung von Protein-Komplexen in<em&gt; Escherichia coli</em&gt; Mit Sequential Peptide Affinity Purification in Kombination mit Tandem-Massenspektrometrie

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida KT2440 and A. chlorophenolicus A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled1.

Freeze-drying is often an easy and convenient way to obtain dry products of viable bacterial cells. An issue of the process is cell survival. We detail here a procedure to investigate how cell survival during freeze-drying is influenced by the properties of the formulation used.

Formulierungen für die Gefriertrocknung von Bakterien und deren Einfluss auf Zellebensdauer

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

High-throughput Reinigung von Affinity-markierter rekombinanter Proteine

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

Reinigung von Transcripts und Metaboliten aus<em&gt; Drosophila</em&gt; Köpfe

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Ein-Schritt-Reinigung von Twin-Strep-markierten Proteine ​​und ihre Komplexe auf Mit Bis (Sulfosuccinimidyl) Strep-Tactin Resin Vernetzte suberat (BS3)

Ein-Schritt-Reinigung von Twin-Strep-markierten Proteine und ihre Komplexe auf Mit Bis (Sulfosuccinimidyl) Strep-Tactin Resin Vernetzte suberat (BS3)