Wir danken Jordan Davis, Yanyan Lin, Eric Schadler und Jimiao Zheng für ihre technische Hilfe zu dieser Studie. Diese Arbeit wurde von der NSF CAREER unterstützt gewähren MCB-1054962 ALA

1. Vorbereiten Große Apfelsaft-Agar-Platten <ol><li> Um 4 Platten zu machen, fügen 375 ml H 2 O, 11,25 g Agar und Fliege einen Rührstab zu einem 1000-ml-Kolben. Dies ist A. Autoklav Mix Mix A mit dem Kolben Deckel lose auf einem 30 min-Sterilisationszyklus für flüssige Güter begrenzt. </li><li> In 125 ml Apfelsaft, 12,5 g Haushaltszucker und einen Rührstab in einen 500-ml-Becherglas. Dies ist Mix B. Wärme Mix B auf einer Plattform unter Rühren erhitzt und die Temperatur bei ungefähr 70 ° C bis zum Autoklavieren der Mischung A ist abgeschlossen. </li><li> Nach Abschluss der Mix Ein Autoklavieren übertragen Mix zu Mix B A. Halten Rühren der kombinierten Mischung vorsichtig und lassen Sie es 15 Minuten abkühlen. Vermeidung von Überkühlung und der Folge Agar Verfestigung vor der Zugabe des Konservierungsmittels. </li><li> Machen Sie 10% (Gewicht / Volumen) Methyl-4-hydroxybenzoat Lösung in Ethanol. Dies wird als Konservierungsmittel dienen. Hinzufügen 3,75 ml der Lösung des oben Apfelsaft-Agar-Mischung. Halten Rühren another 5 min. </li><li> Gießen Sie 125 ml Apfelsaft-Agar-Mischung mit Konservierungsmittel in jeweils 300 cm 2 Kunststoff-oder Styroporplatte. Lassen Sie es auf RT abkühlen und sich verfestigen. Vermeiden Sie die Bildung von Luftblasen beim Gießen. Hinweis: Die Apfelsaft-Agar-Platten sind sofort einsatzbereit. Unbenutzte Platten mit klaren Wraps und lagern bei 4 ° C bis zu einer Woche. </li></ol> 2. Drosophila Embryo Sammlung und Pre-Vernetzungsbehandlung <ol><li> Legen Sie eine Bevölkerung Käfig mit etwa 40.000 bis 50.000 Fliegen aus dem Embryonen werden nicht erhoben. </li><li> Machen Sie zwei Platten, die jeweils 125 ml von Standard-Maismehl-Melassen Medium 12 bestreuen und ca. 4 g getrocknete Bäckerhefe auf jeder Platte. Legen Sie diese Platten in der Bevölkerung Käfig in einem 25 ° C-Inkubator für 48 Stunden, um die Fliegen zu mästen. </li><li> Warm 2 Apfelsaft-Agar-Platten auf 25 ° C Streuen Sie ca. 1 g getrocknete Bäckerhefe auf jedemPlatte. Legen Sie die 2 Platten in der Bevölkerung Käfig zu sammeln 0-1 h alten Embryonen. </li><li> Nehmen Sie die Platten am Ende des 1 h-Sammlung. Ersetzen mit 2 neuen Platten, wenn eine weitere Runde der Sammlung benötigt. </li><li> Fügen Sie genug Wasser, um die Platten bedeckt und resuspendieren Embryonen vorsichtig mit einem feinen Pinsel. Gießen Sie die resuspendiert Embryonen durch eine Zweischicht-Sieb aus Edelstahl-Drahtgewebe. Hinweis: Die oberste Schicht ist von mittlerer Porengröße (850 um), die große Fliegengitterteile sammelt während der Vermietung durch Embryonen hergestellt. Die untere Schicht ist aus feinem mesh (75 um), die Embryonen sammelt während der Vermietung durch Hefe und Wasser hergestellt. </li><li> Verwenden Sie den feinen Pinsel, um die gesammelten Embryonen in einer Sammlung Korb mit einem 50-ml-Tube und feinen Nylongewebe übertragen. Mit Wasser spülen die Embryonen dann kurz trocknen Sie die Masche auf Küchenpapier trocknen. </li><li> Tauchen die Embryonen in 50% Bleichmittel unter leichtem Schwenken für 3 min. Spülen Sie gründlichlich mit Wasser, um alle verbleibenden Bleichmittel entfernen. Tupfen Sie die Maschen auf Küchenpapier trocknen lassen. </li><li> Tauchen Sie die dechorionated Embryonen in Isopropanol unter leichtem Schütteln für 15 Sekunden oder bis zum Zusammenbruch der Multi-Embryo Klumpen (dies sollte nicht länger als 30 Sekunden dauern). Tupfen Sie die Maschen auf Küchenpapier gut trocknen lassen. </li><li> Tauchen die Embryonen in Heptan für 5 min, während mit einer Pipette, Heptan in den Embryonen zu streamen, um sie resuspendierten halten. Tupfen Sie die Maschen auf Küchenpapier trocknen lassen. </li><li> Ströme mit phosphatgepufferter Salzlösung, pH 7,4 (PBS), enthaltend 0,1% Triton X-100 (PBST) für 3 min Spülen der Embryonen. </li><li> Zerlegen Sie den Sammelkorb und die Übertragung der Embryonen zusammen mit dem Mesh zu einem 15-ml-Tube mit 10 ml PBST. Das Röhrchen vorsichtig, um die Embryonen aus dem Netz zu waschen. </li><li> Das Gitter aus der Tube. , Das Glas in einer vertikalen Position und ließ die Embryonen sinken auf den Boden des Rohres durch Schwerkraft. Anmerkung: Etwa 100 bis 300ul von Embryonen für eine 1 Stunde Sammlung von einem Käfig erwartet. </li><li> Entfernen Sie die PBST und weiter zu führen sofort Vernetzung. </li></ol> 3. In vivo Vernetzung der gesammelten Embryonen <ol><li> Bereiten 0,2% Formaldehyd in PBST unmittelbar vor der Verwendung. 10 ml Formaldehyd-Fixierungsmittel, um die Embryonen und Inkubation bei 25 ° C unter leichtem Schütteln auf einem Rundschüttler für 10 min. Nicht verbrauchte Fixierlösung bis zum Ende des Tages. </li><li> Stillen Sie die Vernetzungsreaktion <ol><li> Am Ende der Vernetzung können die Embryonen sinken auf den Boden des Röhrchens. </li><li> Entfernen Sie die Formaldehyd-Lösung. Sofort 10 ml 0,25 M Glycin in PBST hinzufügen, um die Vernetzungsreaktion zu quenchen. Inkubieren bei 25 ° C unter leichtem Schütteln auf einem Rotationsschüttler für 5 min. </li><li> Lassen die Embryonen sinken auf den Boden des Röhrchens. Entfernen Sie den Löschlösung. Mit 10 ml PBST dreimal Waschen der Embryos. </li><li> Vollständig entfernen Sie die PBST Waschlösung mit einer Pipette. Speicher Embryonen bei -80 ° C bis zur Verwendung. </li></ol></li></ol> 4. Protein-Probenvorbereitung und Immunpräzipitation <ol><li> Homogenisieren 500 ul vernetzten Embryonen mit einem Dounce Homogenisator in 2 ml Lyse-Puffer (0,5 M Harnstoff, 0,01% SDS, 2% Triton X-100, 2 mM Phenylmethansulfonylfluorid [PMSF] und Protease-Inhibitor-Cocktail in PBS). Hinweis: Führen Sie dieses und die folgenden Schritte bei RT sofern nicht anders angegeben. </li><li> Transfer des Lysats in 1,5 ml-Zentrifugenröhrchen und schaukeln auf einer rotierenden Plattform 15 Minuten lang, um eine gründliche Lyse sicherzustellen. Zentrifugieren des Lysats bei 16.000 × g für 5 min, um Zelltrümmer zu pelletieren. Speichern Sie den Überstand in ein sauberes 15-ml-Tube. </li><li> 40 &amp;mgr; l Anti-HA-Agarosekügelchen Aufschlämmung in den Überstand und Inkubation auf einer rotierenden Plattform für 2,5 Stunden. </li><li> Pellet die Kügelchen durch Zentrifugation bei 2.500 × g für 5 min. Entfernen Sie den Überstand, aber lassen Sie entximately 250 ul in die Röhre. Resuspendieren der Perlen in der verbleibenden Überstand und Transfer in ein 1,5-ml-Spin-Säule mit 10-um Porenfilter. </li><li> Zentrifugieren Sie die Spin-Säule bei 12.000 g für 10 sec und verwerfen die Strömung durch. </li><li> Waschen der Kügelchen durch Zugabe von 200 ul Waschpuffer (0,1 M Glycin, 1 M NaCl, 1% IGEPAL CA-630, 0,1% Tween-20 in PBS). Zentrifugieren Sie die Spalte bei 12.000 g für 10 sec und entsorgen Sie die Wäsche. Wiederholen Sie den Wasch zwei weitere Male. </li><li> 40 &amp;mgr; l Elutionspuffer (2 mg / ml HA-Peptid in PBS) zu den Kügelchen und Inkubation bei 37 ° C für 15 min unter leichtem Schütteln alle 5 min. Zentrifugieren Sie die Spalte bei 12.000 g für 10 sec und sammeln das Eluat. </li><li> Elution des Komplexes durch Western-Blot-Analyse Bestätigen Sie mit Anti-HA-13. </li></ol>

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A., Lasko, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fat+facets+interacts+with+vasa+in+the+Drosophila+pole+plasm+and+protects+it+from+degradation.">Fat facets interacts with vasa in the Drosophila pole plasm and protects it from degradation.</a> Current biology : CB. 13, 1905-1909 (2003).</li></ol>

Die Autoren haben nichts zu offenbaren.

Formaldehyd wurde üblicherweise als Vernetzungsmittel für die Identifizierung von Protein-Protein-und Protein-Nukleinsäure-Wechselwirkungen verwendet. Seine gute Löslichkeit und Permeabilität der Zellmembran, zusammen mit der Kompatibilität mit nachgeschalteter Massenspektrometrie Verfahren machen Formaldehyd ein idealer Kandidat für die intrazelluläre Vernetzungsmittel Anwendungen 3,15-17. Insbesondere wurde es erfolgreich eingesetzt, um mRNAs mit Vasa, einen kritischen Keimzelle RNA-Helikase in ...

Isolierung von Protein aus mehreren Unterbaugruppen und DNA-oder RNA-Protein-Komplexen durchgeführt, um Proteinkomplexe, genomische Loci von DNA-bindenden regulatorischen Proteine ​​oder RNA-Targets von RNA-bindenden Proteinen erkannt identifizieren. Verschiedene Methoden ermöglichen genomweiten Identifizierung von DNA-Stellen von Transkriptionsfaktoren oder Chromatin-Proteine ​​(ChIP-Seq) 1 und RNA-Targets mit einer bestimmten RNA-bindenden Protein (CLIP-Seq) 2 zugeordnet anerkannt. Die Bibliotheken der RNA-oder DNA-cDNAs abgeleiteten Ziele werden dann tief sequenziert. Diese Verfahren verwenden chemische oder UV-induzierte Vernetzung, um die Komplexe, gefolgt von Immunopräzipitation (IP) mit einem Antikörper gegen ein Protein-Komponente des Komplexes untersucht stabilisieren. Während der Entwicklung eines Organismus bilden viele Proteinkomplexe transient. Daher ist es von entscheidender Bedeutung zur Analyse der Zusammensetzung und Funktion dieser Komplexe in vivo, die molekularen Mechanismen zu verstehen, die Entwickler zu steuernelopment. Solch ein in vivo-Analyse überlegen wäre in vitro-Ansatz, da es praktisch unmöglich ist, nativen Konzentrationen der zusammenwirkenden Komponenten und zelluläre biochemische Umgebung in vitro zu reproduzieren. Hier zeigen wir eine in vivo-Ansatz, den wir verwenden, erfolgreich auf großen Proteinkomplexen von Drosophila-Embryonen zu isolieren. In diesem Verfahren werden Protein-Komplexe in den lebenden Embryonen mit einer niedrigen Konzentration von Formaldehyd und anschließender Proteinkomplexe von Interesse werden von IP mit einem Antikörper isoliert gegen eine bekannte Komponente des Komplexes, gefolgt von der Gelreinigung der Komplexe und Massenspektrometrie-Analyse zu vernetzenden identifizieren unbekannt komplexe Bauteile. Da Formaldehyd in der Lage ist, die Zellmembran zu durchdringen und eine Vernetzungs Bereich von 2,3-2,7 Å 3, können Protein-Komplexen in vivo, vernetzt werden und die komplexen Bauteilen, die nahe zueinander zu sein. In diesem Artikel werden wirbeschreiben dieses Verfahren mit der Isolierung von Tudor (TUD) Proteinkomplex als Beispiel. Tud ist eine Keimbahn-Protein, die für die Entwicklung der Keimbahn 4-7 ist. Dieses Protein enthält 11 Tud Domains bekannt, mit methylierte Arginine Lysine oder von anderen Polypeptiden 8-10 interagieren. Früher haben wir eine transgene Drosophila-Linie, die HA-markierte Funktions Tud 5 drückt, und daher wird spezifische Anti-HA-Antikörper verwendet, um komplexe Tud nach Vernetzung Pulldown generiert haben. Zusätzlich zu Protein-Protein-Quervernetzungen können Formaldehyd Nukleinsäure-Protein-Vernetzungen zu erzeugen, und wird in der Chip-seq Experimente verwendet. Ferner ist in Drosophila, in vivo Vernetzung mit Formaldehyd hat die Identifizierung eines RNA-Ziel-RNA-Helicase-Protein Vasa 11 erlaubt. Während in diesem Artikel beschreiben wir ein Verfahren für die In-vivo-und Vernetzungs purifizierung von Proteinkomplexen von Drosophila-Embryonen, kann dieses Verfahren für andere Organismen und Gewebe angepasst werden.

<table border="0" cellpadding="0" cellspacing="0" width="858">
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Drosophila agar</td>
			<td style="width:235px;">Lab Scientific (http://www.labscientific.com/)</td>
			<td style="width:119px;">FLY-8020-1</td>
			<td style="width:289px;">Do not autoclave the water and agar mix for prolonged period of time as it will cause the apple juice plates to become fragile</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Methyl 4-hydroxybenzoate</td>
			<td style="width:235px;">Sigma (http://www.sigmaaldrich.com/united-states.html)</td>
			<td style="width:119px;">H5501</td>
			<td style="width:289px;">Other name: TEGOSEPT</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Population cage</td>
			<td style="width:235px;">Flystuff (http://www.flystuff.com/)</td>
			<td style="width:119px;">59-104</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Fine nylon mesh</td>
			<td style="width:235px;">Flystuff (http://www.flystuff.com/)</td>
			<td style="width:119px;">57-102</td>
			<td style="width:289px;">When making the collection basket, cut the mesh slightly larger than the opening of the falcon tube cap to ensure a tight seal</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dounce homogenizer</td>
			<td style="width:235px;">Sigma (http://www.sigmaaldrich.com/united-states.html)</td>
			<td style="width:119px;">D8938-1SET</td>
			<td style="width:289px;">Chill the homogenizer on ice and prerinse with cold lysis buffer before use to prevent protein degradation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Protease inhibitor cocktail&#160;</td>
			<td style="width:235px;">Roche (http://www.rocheusa.com/portal/usa)</td>
			<td style="width:119px;">4693132001</td>
			<td style="width:289px;">PBS could be used to prepare concentrated protease inhibitor stock solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-HA agarose beads</td>
			<td style="width:235px;">MBL international (http://www.mblintl.com/)</td>
			<td style="width:119px;">561-8</td>
			<td style="width:289px;">The kit also includes HA-peptide and spin columns</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HA-peptide</td>
			<td style="width:235px;">MBL international (http://www.mblintl.com/)</td>
			<td style="width:119px;">561-8</td>
			<td style="width:289px;">Prepare to 2 mg/ml with PBS&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spin Column</td>
			<td style="width:235px;">MBL international (http://www.mblintl.com/)</td>
			<td style="width:119px;">561-8</td>
			<td style="width:289px;">Spin columns are included as part of the kit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Isopropanol</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">BP26324</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Triton X-100</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">BP151-500</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Heptane</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">H350-4</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">PBS</td>
			<td style="width:235px;">Invitrogen (https://www.lifetechnologies.com/us/en/home.html)</td>
			<td style="width:119px;">AM9625</td>
			<td style="width:289px;">Dilute from 10 X to 1 X with nanopure water before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Formaldehyde</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">BP531-500</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycine</td>
			<td style="width:235px;">BioRad (www.bio-rad.com/&#8206;)</td>
			<td style="width:119px;">161-0724</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SDS</td>
			<td style="width:235px;">BioRad (www.bio-rad.com/&#8206;)</td>
			<td style="width:119px;">161-0301</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Urea</td>
			<td style="width:235px;">BioRad (www.bio-rad.com/&#8206;)</td>
			<td style="width:119px;">161-0730</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Phenylmethanesulfonyl fluoride</td>
			<td style="width:235px;">Sigma (http://www.sigmaaldrich.com/united-states.html)</td>
			<td style="width:119px;">P7626-1G</td>
			<td style="width:289px;">Prepare 200 mM stock solution in isopropanol then dilute to working concentration of 2 mM in lysis buffer</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">IGEPAL CA-630</td>
			<td style="width:235px;">Sigma (http://www.sigmaaldrich.com/united-states.html)</td>
			<td style="width:119px;">I8896-50ML</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tween 20</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">BP337-100</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">15-ml tubes</td>
			<td style="width:235px;">USA Scientific (http://www.usascientific.com/)</td>
			<td style="width:119px;">1475-0511</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bleach</td>
			<td style="width:235px;">Clorox brand</td>
			<td style="width:119px;"></td>
			<td style="width:289px;">Dilute with equal volume of nanopure water to make 50% bleach</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">50-ml tubes</td>
			<td style="width:235px;">BD Biosciences (http://www.bdbiosciences.com/home.jsp)</td>
			<td style="width:119px;">352098</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Top layer sieve</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">04-884-1AK</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Bottom layer sieve</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">04-884-1BA</td>
			<td style="width:289px;"></td>
		</tr>
	</tbody>
</table>

an in vivo crosslinking approach to isolate protein complexes from drosophila embryos

Viele zelluläre Prozesse werden von mehreren Unterproteinkomplexe gesteuert. Häufig werden diese Komplexe zu bilden vorübergehend und erfordern nativen Umgebung zu montieren. Deshalb, um diese funktionellen Protein-Komplexe zu identifizieren, ist es wichtig, sie in vivo vor der Zell-Lyse und anschließender Reinigung stabilisieren. Hier beschreiben wir eine Methode verwendet, um große Bona-fide-Protein-Komplexe von Drosophila-Embryonen zu isolieren. Dieses Verfahren wird auf den Embryo Permeabilisierung und Stabilisierung der Komplexe in den Embryonen von in vivo Vernetzung unter Verwendung einer niedrigen Konzentration an Formaldehyd, die leicht überqueren die Zellmembran beruht. Anschließend wird die Proteinkomplex von Interesse immungereinigt, gefolgt von der Gelreinigung und durch Massenspektrometrie analysiert. Wir veranschaulichen dieses Verfahren unter Verwendung eines Reinigungs Tudor-Protein-Komplex, die für die Keimbahn-Entwicklung ist. Tudor ist ein großes Protein, das mehrere Tudor-Domänen enthält - kleinModule, die mit methylierte Lysine oder Arginine von Zielproteinen wechselwirken. Diese Methode kann für die Isolierung von nativen Proteinkomplexe aus verschiedenen Organismen und Geweben angepasst werden.

Schritt 7% Gel (in 1 dargestellt), gefolgt von Western-Blot (Fig. 2) - die Wirksamkeit der Vernetzung und die erfolgreiche Reinigung des vernetzten Tud Protein-Komplex wurden durch SDS-PAGE auf einem 3% analysiert. Der Zweck der Verwendung von 3% - 7% Schritt Gel auf die wirksame Trennung von vernetzten Tud Proteinkomplex aus dem verbleibenden unvernetzten Tud Protein und Konzentration des Komplexes beruht. Unter unseren oben erwähnten in vivo Vern...

Watch this Scientific Journal Video about An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos at JoVE.com

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Mehrkomponenten-Protein-Komplexe spielen eine entscheidende Rolle bei der zellulären Funktion und Entwicklung. Hier beschreiben wir ein Verfahren verwendet, um native Protein-Komplexe von Drosophila-Embryonen isoliert nach in vivo Vernetzung durch Reinigung der vernetzten Komplexe für die nachfolgende Struktur-Funktions-Analyse.

Ein In vivo Vernetzung Ansatz zur Proteinkomplexe Von Isolieren Drosophila Embryonen

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

an-vivo-crosslinking-approach-to-isolate-protein-complexes-from

Research

JoVE Journal

Biology

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

16.6K Aufrufe.  Murray State University.  Mehrkomponenten-Protein-Komplexe spielen eine entscheidende Rolle bei der zellulären Funktion und Entwicklung. Hier beschreiben wir ein Verfahren verwendet, um native Protein-Komplexe von Drosophila-Embryonen isoliert nach in vivo Vernetzung durch Reinigung der vernetzten Komplexe für die nachfolgende Struktur-Funktions-Analyse. 

Serie: An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Vorbereitung der neuronalen Kulturen aus Midgastrula Bühne Drosophila Embryonen

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Forschung

Biologie

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated through myosin-actin interactions and play an important role in our physiology.  During development, they enable cells to move from one location to the next in order to form the early structures of tissue.  Traction forces help in the healing processes.  They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body.  These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor.  The most common method by which to study cells in vitro has been to use a glass or polystyrene dish.  However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces.  Our lab has developed a technique to overcome these limitations.  The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process.  The pillars we use are 3 &mu;m in diameter, 10 &mu;m tall, and are configured in a regular array with 9 &mu;m center-to-center spacing.  But these physical dimensions can be readily varied to accommodate a variety of studies.  We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS.  We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections.  We call these substrates microfabricated post-array-detectors, or mPADs.  Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.

Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces

In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility.

Mikrofabrizierten Post-Array-Detektoren (mPADs): Ein Ansatz zur Mechanical Forces Isolate

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Vorbereitung der Embryonen für Elektronenmikroskopie der Drosophila Embryonale Herz Rohr

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

Isolierung von Labile Multi-Protein-Komplexe durch In vivo Kontrollierte Cellular Cross-Linking und Immuno-magnetische Affinitätschromatographie

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

In-vivo-Zentrifugation von Drosophila Embryonen

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

Imaging Protein-Protein-Wechselwirkungen In vivo</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

Upright Imaging von Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Primären Zellkulturen aus Drosophila Gastrula Embryonen

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

Imaging Zellgröße Veränderung der Lebens- Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Isolierung und Reinigung von Kinesin aus Drosophila Embryonen

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...