This work was supported by a grant from the Canadian Institutes for Health Research (MOP-86514).

Experimentelle Verfahren mit Mäusen wurden von der Animal Care Committee der McGill University (Protokoll 4966) zugelassen. 1. Herstellung von Proteinextrakten aus kultivierten Zellen <ol><li> Waschen kultivierten Zellen zweimal mit 10 ml eiskalter phosphatgepufferter Salzlösung (PBS). </li><li> Kratzen Sie anhaftenden Zellen entweder mit einem Gummiwischer oder einem Kunststoffzellschaber in 1 ml eiskaltem PBS, Transfer Suspension in ein 1,5 ml Mikrozentrifugenröhrchen. </li><li> In einer Mikrozentrifuge für 5 Minuten bei 700 × g bei 4 ° C. Absaugen PBS. </li><li> 100 l eiskaltem Zytoplasma-Lysepuffer (Tabelle 1) pro 10 7 Zellen und Pipette auf und ab. </li><li> Inkubieren auf Eis für 20 min. </li><li> Spin für 10 min bei voller Geschwindigkeit in einer Mikrozentrifuge bei 4 ° C. </li><li> Verwerfen Pellet. Überstand in neue 1,5 ml Mikrozentrifugenröhrchen und auf Eis halten. </li><li> DetHermelins Proteinkonzentration (gewöhnlich 1 - 10 &amp; mgr; g / &amp; mgr; l) unter Verwendung des Bradford-Assay 23. </li><li> Aliquot und Speichern von Zellextrakten bei -80 ° C bis zur Verwendung. </li></ol> 2. Herstellung von Proteinextrakten aus Maus Leber und Milz <ol><li> Euthanize eine Maus mit CO 2 Inhalation. </li><li> Legen Sie das Tier eingeschläfert auf eine saubere Unterlage über einen Seziersaal Bord. Öffnen Sie den Unterleib mit einer Schere. </li><li> Präparieren Sie die Leber und die Milz mit einer Schere und Pinzette, und spülen Sie jedes Gewebe in ca. 50 ml eiskaltem PBS. </li><li> Unmittelbar geschnitten Gewebe in kleine Stücke mit einem Skalpell (zB etwa 1-2 mm 3). </li><li> Unverzüglich, legte Stücke von Gewebe in einem frischen Kryoröhrchen und Schnapp frieren sie in flüssigem Stickstoff dann. Shop schockgefroren Gewebe Aliquots bei -80 ° C bis zur Verwendung. </li><li> Homogenisieren einem Stück gefrorenes Gewebe (ungefähr 1 bis 2 mm 3) in 0,25 bis 0,5 mleiskaltem zytoplasmatischen Lysepuffer (Tabelle 1) mit einem Gewebe-Homogenisator 10 sec. </li><li> Übertragen Homogenat 1,5 ml Mikrozentrifugenröhrchen und Chill auf Eis für 20 min. </li><li> Spin für 10 min bei voller Geschwindigkeit in einer Mikrozentrifuge bei 4 ° C. </li><li> Entsorgen Pellet und Überstand in neues 1,5-ml-Mikrozentrifugenröhrchen. Halten Sie auf dem Eis. </li><li> Bestimmen Proteinkonzentration (gewöhnlich 1 - 10 &amp; mgr; g / &amp; mgr; l) unter Verwendung des Bradford-Assay 23. </li><li> Aliquot und Speichern von Zellextrakten bei -80 ° C bis zur Verwendung. </li></ol> 3. Herstellung von radioaktiv markierten IRE-Sonde <ol><li> Linearisieren des IRE-haltigen Plasmid I-12.CAT 22 durch Inkubation bei 37 ° C für 1 Stunde mit der Restriktionsendonuklease XbaI (1 U pro ug Plasmid), spalt hinter der IRE-Sequenz. Das linearisierte Plasmid wird als Matrize für die in vitro-Transkription verwendet werden. </li><li> Richten Sie einn in vitro-Transkriptions-Reaktion in einem Gesamtvolumen von 20 ul. Verwenden Sie die in Tabelle 2 angegebenen Stammlösungen und hinzuzufügen: 1 ul linearisierte Plasmid-Vorlage, 4 ul Transkriptionspuffer; 1 &amp; mgr; l Mischung aus ATP / CTP / GTP-Mix, 10 &amp; mgr; l [α- 32 P] -UTP, 2 &amp; mgr; l Dithiothreitol, 1 ul RNase-Inhibitor und 1 ul T7 RNA-Polymerase. Mischen Sie durch Auf- und Abpipettieren. </li><li> Inkubiere bei 40 ° C für 1 h 24. </li></ol> 4. Reinigung von radioaktiv markierten IRE-Sonde <ol><li> Enden in vitro-Transkriptions-Reaktion durch Zugabe von 1 ul 0,5 M EDTA, pH 8 Mix durch Auf- und Abpipettieren. </li><li> Zugabe von 10 ul 10 mg / ml tRNA, die als Träger für eine bessere Niederschlag. Mischen Sie durch Auf- und Abpipettieren. </li><li> Hinzufügen 82,5 ul 3 M Ammoniumacetat. Durch Vortexen mischen. </li><li> Hinzufügen 273 ul Ethanol. Durch Vortexen mischen. </li><li> Stehen lassen bei Raumtemperatur für 5 min. </li><li> Spin für 10 Minuten bei voller Geschwindigkeit in einer Mikrozentrifuge bei RT. Überstand verwerfen. </li><li> Waschen Sie das Pellet mit 100 ul 70% Ethanol. </li><li> Spin für 10 Minuten bei voller Geschwindigkeit in einer Mikrozentrifuge bei RT. Überstand verwerfen. </li><li> Luft trocknen Pellet für 10 min. </li><li> Pellet in 100 &amp; mgr; l doppelt destilliertes, zuvor autoklaviert H 2 O. </li><li> Quantifizierung der Radioaktivität in einem Flüssigszintillationszähler, aliquoten radioaktiv markierten IRE Sonde und bei -80 ° C bis zur Verwendung. Gefrorene Aliquots für 3 Wochen verwendet werden. </li></ol> 5. Herstellung von einem nativen Polyacrylamidgel für die EMSA <ol><li> Montieren Sie das Gel (16 x 16 cm) unter Verwendung von 1,5 mm Abstandshalter und Kamm. </li><li> Um eine 6% nativen Polyacrylamidgel vorzubereiten, mit den in Tabelle 3 gezeigten Stammlösungen mischen 7,5 ml 40% Acrylamid. Bisacrylamid, 5 ml 5 x TBE und 37,5 mldoppelt destilliertem H 2 O. </li><li> 0,5 ml von 10% frisch zubereitet Ammoniumpersulfat (APS) und 25 ul Tetramethylethylendiamin (TEMED). </li><li> Gießen Sie sofort die Acrylamidlösung zum Gel und lassen Sie es zu polymerisieren. Warten für ungefähr 30 min. </li><li> Montieren Sie die Elektrophorese-Vorrichtung mit dem Gel, füllen Sie die Tanks mit 0,5x TBE und eine Verbindung mit der Stromversorgung. </li></ol> 6. Electrophoretic Mobility Shift Assay <ol><li> Verdünne 25 &amp; mgr; g Proteinextrakt aus Zellen oder Geweben mit der zytoplasmatischen Lysepuffer (Tabelle 1) in einem Gesamtvolumen von 10 ul (niedrigeren Proteinkonzentrationen können auch angewandt werden). Halten Sie auf dem Eis. Falls erforderlich, fügen Sie 1 ul 1: 4 verdünnt 2-Mercaptoethanol (2-ME) zum ruhenden IRP1 (Endkonzentration: 2%) aktivieren 25. </li><li> Verdünnen Sie die radioaktiv markierten Sonde IRE in doppelt destilliertem H 2 O zu 200.000 cpm / ul, Hitze denaturieren bei 95 °C für 1 min und Abkühlen bei Raumtemperatur für mindestens 5 min. </li><li> Bis ein EMSA Reaktion durch Zugabe von 1 ul radioaktiv markierten Sonde an die IRE Proteinextrakt ein. </li><li> Inkubieren für 20 min bei RT. </li><li> 1 ul 50 mg / ml Heparin zu dem Reaktions (um nicht-spezifische Protein-Wechselwirkungen mit der Sonde 9 inhibieren) angeschlossen und die Inkubation für weitere 10 min. Aliquot der Stammlösung von Heparin (50 mg / ml) und bei -80 ° C. </li><li> Wenn lange radiomarkierten Sonden (&gt; 60 Nukleotide), Zugabe von 1 &amp; mgr; l RNase T1 (1 U / ul) und Inkubation für 10 min bei RT, um unspezifische Proteinbindung zu reduzieren, um die Sonde und eine bessere Trennung des RNA / Protein ermöglichen Komplex während der Elektrophorese. </li><li> In 3 ul Auftragspuffer (80% Glycerin + Bromphenolblau), mischen und Last auf der 6% nicht-denaturierenden Polyacrylamidgel. </li><li> Führen Sie das Gel für 60 min bei 130 V (5 V / cm). </li><li> Transfer das Gel auf eine große Filterpapier und trocken. </li><li> Expose an einen Film und entwickeln Autoradiographie. Belichtungszeit kann von 1 Stunde (oder weniger) bis O / N liegen. </li></ol>

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The authors declare that they have no competing financial interests.

Hier beschreiben wir ein Protokoll, das entwickelt wurde, um die IRE-Bindungsaktivitäten von IRP1 und irp2 studieren, und wir zeigen repräsentative Daten. Durch die Verwendung von verschiedenen Sonden, kann dieses Protokoll auch für die Untersuchung von anderen RNA-bindenden Proteinen eingestellt werden. Ein kritischer Schritt ist die Größe der Sonde. Verwendung von langen Sonden, die gemeinsam ist, wenn die genaue Bindungsstelle nicht bekannt ist, kann in der RNA / Protein-Komplexe, die nicht anders migrieren als ...

Die EMSA wurde ursprünglich entwickelt, um die Assoziation von DNA-bindenden Proteinen mit DNA-Zielsequenzen 1,2 studieren. Das Prinzip ist ähnlich für RNA / Protein-Interaktionen 3, die im Mittelpunkt dieses Artikels ist. Kurz gesagt wird RNA negativ geladen und werden in Richtung der Anode während der nicht-denaturierenden Elektrophorese in Polyacrylamid (oder Agarose) Gelen wandern. Migration innerhalb des Gels hängt von der Größe der RNA, die proportional zu ihrer Ladung ist. Spezifische Bindung eines Proteins an RNA verändert seine Mobilität und der Komplex wandert langsamer als das freie RNA. Dies ist vor allem auf eine Erhöhung der Molekularmasse, sondern auch Änderungen in der Ladung und ggf. Konformation. Mit Hilfe eines markierten RNA als Sonde ermöglicht eine einfache Überwachung des &quot;Gelretardation&quot; oder &quot;Bandshift&quot;. Verwendung von 32 P-markierten RNA-Sonden ist sehr verbreitet und bietet hohe Empfindlichkeit. Die Migration von RNA / Protein-Komplexen und freien RNA nachgewiesen werdendurch Autoradiographie. Nachteile sind die kurze Halbwertszeit von 32 P (14,29 Tage), die allmähliche Verschlechterung der Qualität der Sonde durch Radiolyse, das Erfordernis einer Radioaktivität Lizenz und die Infrastruktur für die Radioaktivität der Arbeit, und potentielle biologische Sicherheit betrifft. Daher alternative nichtisotopische Methoden zur Markierung der RNA-Sonde entwickelt, zum Beispiel mit Fluorophoren oder Biotin, der Nachweis durch Fluoreszenz- oder Chemilumineszenz-Abbildungs ​​4,5 ermöglichen. Beschränkungen dieser Verfahren sind die höheren Kosten und oft eine verringerte Empfindlichkeit gegenüber Isotopenmarkierung, und das Potential des nicht-isotopische Markierungen, mit der RNA / Protein-Wechselwirkung stören. Nicht-denaturierenden Polyacrylamid-Gelen sind für die meisten Anwendungen EMSA und werden häufig verwendet. Gelegentlich kann Agarosegelen eine Alternative für die Analyse von großen Komplexen darstellen. Der große Vorteil der EMSA ist, dass es kombiniert Einfachheit, Sensitivität und Robustheit 4 &lt;/ Sup&gt;. Der Test kann innerhalb weniger Stunden abgeschlossen werden kann und keine ausgeklügelte Instrumentierung erforderlich. RNA / Protein-Interaktionen können von der EMSA bei Konzentrationen von nur 0,1 nM oder weniger erkannt werden, und in einem weiten Bereich von Bindungsbedingungen (pH 4,0 bis 9,5, monovalente Salzkonzentration von 1 bis 300 mM, und die Temperatur 0 - 60 ° C). RNA / Protein-Komplexbildung kann ebenfalls durch den Filter-Bindungsassay untersucht werden. Dies ist ein einfaches, schnelles und kostengünstiges Verfahren auf der Basis der Beibehaltung der RNA / Protein-Komplexen in einem Nitrocellulose-Filter, während eine freie RNA Sonde durch 6 durchläuft. Im Vergleich zu EMSA, ist es in Fällen, wo die RNA-Sonde enthält mehrere Bindungsstellen oder der Rohextrakt enthält mehrere RNA-Bindeproteine, die an die Sonde binden, an der gleichen Stelle begrenzt ist. Während mehrere RNA / Protein-Interaktionen werden Erkennung durch den Filter-Bindungstest entgehen, können sie leicht durch EMSA visualisiert werden. In einigen Fällen ist die Visualisierung Vorabendn möglich, wenn zwei RNA / Protein-Komplexe co-migrieren (zum Beispiel menschliche IRP1 / IRE und irp2 / IRE-Komplexe), durch Zugabe eines Antikörpers gegen eines der RNA-Bindeproteine ​​zu der EMSA Reaktion, wodurch man weitere Retardierung auf dem Gel ( &quot;Supershift&quot;) 7. Die EMSA wurde weithin verwendet, um IRP1 und irp2, die nach der Transkriptionsregulatoren des Eisenstoffwechsels 8-10 sind zu studieren. Sie funktionieren über die Bindung an IREs phylogenetisch konservierten Haarnadelstrukturen innerhalb der UTRs mehrerer mRNAs 11. IREs wurden zuerst in den mRNAs Ferritin 12 und Transferrin-Rezeptor 1 (TfR1) 13, Proteinen von Eisen Lagerung und Aufnahme beziehungsweise kodiert entdeckt. Später IREs wurden in erythroiden spezifische Aminolaevulinat Synthase (ALAS2) 14, mitochondrialen Aconitase 15. Ferroportin 16, zweiwertige Metall Transporter 1 (DMT1) 17, Hypoxie-induzierbaren Faktors 2 gefunden <em&gt; α (HIF2α) 18 und anderen mRNAs 19-21. Der Prototyp H- und L-Ferritin-mRNAs enthalten ein IRE in ihren 5&#39;-UTR, während TfR1 mRNA enthält mehrere IREs in ihrem 3&#39;-UTR. IRE / IRP Wechselwirkungen spezifisch hemmen Ferritin-mRNA-Translation durch sterisch Sperrung der Assoziation der 43S ribosomalen Untereinheit; Darüber hinaus stabilisieren sie TfR1 mRNA gegen endonukleolytische Spaltung. IRP1 und irp2 Aktien umfassende Sequenzähnlichkeit und weisen eine hohe IRE-Bindungsaktivität in eisenversorgten Zellen. In eisenversorgten Zellen versammelt IRP1 eine Cuban Fe-S-Cluster, der es um cytosolische Aconitase wandelt auf Kosten der IRE-Bindungsaktivität, während irp2 erfährt proteasomalen Abbau. Somit hängen die IRE / IRP Wechselwirkung bei der zellulären Eisenstatus, sondern auch durch andere Signale, wie zum Beispiel H 2 O 2, Stickstoffmonoxid (NO) oder Hypoxie. Hier beschreiben wir das Protokoll für die Beurteilung der IRE-Bindungsaktivität von Rohöl Zell- und Gewebeextrakten von EMSA. Wir verwendeten eine 32 P-markiertem H-Ferritin-IRE-Sonde, die durch in vitro-Transkription von Plasmid-DNA-Matrize (I-12.CAT), wobei der IRE-Sequenz wurde ursprünglich in Sense-Orientierung stromabwärts des T7-RNA-Polymerase-Stelle durch eingeführten generated Klonen von geglüht synthetische Oligonukleotide 22.

<table border="0" cellpadding="0" cellspacing="0" width="430">
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		<tr height="17">
			<td height="17" style="height:17px;width:195px;">Name of the Materials&#160;</td>
			<td style="width:119px;">Company</td>
			<td style="width:117px;">Catalog Number&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">leupeptin</td>
			<td>SIGMA</td>
			<td>L2884</td>
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			<td height="17" style="height:17px;">PMSF</td>
			<td>SIGMA</td>
			<td align="right">78830</td>
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			<td height="17" style="height:17px;">BioRad Protein Assay</td>
			<td>BIORAD</td>
			<td>500-0006</td>
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		<tr height="17">
			<td height="17" style="height:17px;">T7 RNA polymerase</td>
			<td>Thermoscientific</td>
			<td>EPO111</td>
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		<tr height="17">
			<td height="17" style="height:17px;">Rnase Inhibitor</td>
			<td>Invitrogen</td>
			<td>15518-012</td>
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		<tr height="17">
			<td height="17" style="height:17px;">UTP [alpha-32P]</td>
			<td>Perkin-Elmer</td>
			<td>NEG507H</td>
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		<tr height="17">
			<td height="17" style="height:17px;">Scintillation liquid</td>
			<td>Beckman Coulter</td>
			<td align="right">141349</td>
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		<tr height="17">
			<td height="17" style="height:17px;">heparin</td>
			<td>SIGMA</td>
			<td>H0777</td>
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		<tr height="17">
			<td height="17" style="height:17px;">Rnase T1</td>
			<td>Thermoscientific</td>
			<td>EN0541</td>
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		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
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			<td height="17" style="height:17px;">Name of the Equipments&#160;</td>
			<td>Company</td>
			<td>Catalog Number&#160;</td>
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			<td height="17" style="height:17px;">Tissue Ruptor</td>
			<td>Qiagen</td>
			<td align="right">9001271</td>
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		<tr height="17">
			<td height="17" style="height:17px;">Scintillation counter</td>
			<td>Beckman Coulter</td>
			<td>LS6500</td>
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		<tr height="17">
			<td height="17" style="height:17px;">Protean II xi Cell&#160;&#160;</td>
			<td>BIORAD</td>
			<td>165-1834</td>
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		<tr height="17">
			<td height="17" style="height:17px;">20 wells combs 1.5mm thick</td>
			<td>BIORAD</td>
			<td>165-1868</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1.5 mm spacers</td>
			<td>BIORAD</td>
			<td>165-1849</td>
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		<tr height="17">
			<td height="17" style="height:17px;">PowerPac</td>
			<td>BIORAD</td>
			<td>164-5070</td>
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electrophoretic mobility shift assay (emsa) for the study of rna-protein interactions: the ire/irp example

RNA / Protein-Interaktionen sind entscheidend für die Post-Transkriptionsregulationswege. Unter den am besten charakterisierten zytosolische RNA-bindende Proteine ​​sind Eisen regulatorischen Proteinen, IRP1 und irp2. Sie binden an responsive Elemente (IRES) innerhalb der untranslatierten Regionen (UTR) von mehreren Ziel-mRNAs bügeln, damit die mRNA Translation oder Stabilität Steuerung. IRE / IRP Wechselwirkungen wurden weitgehend von der EMSA untersucht. Hier beschreiben wir die EMSA-Protokoll für die Analyse der IRE-Bindungsaktivität von IRP1 und irp2, die generali können, um die Aktivität von anderen RNA-bindenden Proteinen und zu beurteilen. A Rohprotein Lysat eine RNA-Bindungsprotein, oder ein gereinigtes Präparat des Proteins enthält, wird mit einem Überschuss von 32 P-markierten RNA-Sonde inkubiert, wobei zur Komplexbildung. Heparin zugesetzt wird, um nicht-spezifische Protein ausschließen zu Sondenbindung. Anschließend wird die Mischung durch nicht-denaturierende Elektrophorese auf einem Polyacrylamidgel analysiert. Die freien Sondewandert schnell, während die RNA / Proteinkomplex Exponate verzögert Mobilität; daher wird das Verfahren auch als &quot;Gelretardation&quot; oder &quot;Bandshift&quot; -Assay. Nach Beendigung der Elektrophorese wird das Gel getrocknet und RNA / Protein-Komplexe, sowie freier Sonde durch Autoradiographie detektiert. Das übergeordnete Ziel des Protokolls ist es, nachzuweisen und zu quantifizieren IRE / IRP und andere RNA / Protein-Interaktionen. Außerdem kann EMSA verwendet, um die Spezifität, Affinität und Stöchiometrie des RNA / Protein-Wechselwirkung untersucht bestimmen.

Ein radioaktiv markiertes IRE Sonde hergestellt wurde, wie in den Abschnitten 3 und 4 des Protokolls beschrieben. Die Sequenz der Sonde war 5&#39;-GGGCGAAUUC GAGCUCGGUA CCCGGGGAUC CUG C UUCAA C AGUGC UUGGA CGGAUCCU-3 &#39;; die fettgedruckten Nukleotide stellen einen ungepaarten C-Rest und die Schleife, die kritische IRE Merkmale sind. Die spezifische Radioaktivität der Probe betrug 4,5 x 10 &amp; sup9; cpm / ug RNA. Um die Auswirkungen von Stör...

Watch this Scientific Journal Video about Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example at JoVE.com

Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Hier präsentieren wir ein Protokoll zur RNA / Protein-Interaktionen zu untersuchen. Elektrophoretische Mobilitäts-Shift-Assay (EMSA) auf der differentielle Migration RNA / Protein-Komplexe und freie RNA während nativer Gelelektrophorese. Durch Verwendung eines radioaktiv markierten RNA-Sonde kann RNA / Protein-Komplexe durch Autoradiographie visualisiert werden.

Electrophoretic Mobility Shift Assay (EMSA) für das Studium von RNA-Protein-Wechselwirkungen: Die IRE / IRP Beispiel

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron ...

electrophoretic-mobility-shift-assay-emsa-for-study-rna-protein

Research

JoVE Journal

Biology

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

53.3K Aufrufe.  Jewish General Hospital.  Hier präsentieren wir ein Protokoll zur RNA / Protein-Interaktionen zu untersuchen. Elektrophoretische Mobilitäts-Shift-Assay (EMSA) auf der differentielle Migration RNA / Protein-Komplexe und freie RNA während nativer Gelelektrophorese. Durch Verwendung eines radioaktiv markierten RNA-Sonde kann RNA / Protein-Komplexe durch Autoradiographie visualisiert werden. 

Serie: Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testen visueller Sensibilität für die Geschwindigkeit und Richtung der Bewegung in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Forschung

Biologie

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

Optimierte Fibringel Bead Assay für das Studium der Angiogenese

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Computer-Generated-Tiermodell Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Do-It-Yourself Einrichtung zur Rückgewinnung von kryokonservierten Proben versehentlich fallengelassen in Kryo-Lagerbehälter

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

Eine flüssige Phase Affinity Capture Assay Verwendung von magnetischen Kügelchen, um Protein-Protein Interaktion Studie: Das Poliovirus-Nanobody Beispiel

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Die MultiBac Protein Complex Produktion Platform am EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

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 ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

Die Kombination von Einzelmolekül-Imaging und Manipulation für das Studium der Protein-DNA-Wechselwirkungen

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Chromatinimmunpräzipitation Assay zur Identifizierung von Arabidopsis-Protein-DNA-Wechselwirkungen<em&gt; In-vivo-</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

Screening für Funktionelle nichtkodierenden genetischen Varianten Mit elektrophoretische Mobilitäts-Shift-Assay (EMSA) und DNA-Affinitätspräzipitation Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical ...