Wir danken der O&#39;Bryan Labor an UIC für kritische experimentelle Evaluation und Diskussion. UIC Zentrum für Klinische und Translational Sciences Zuschuss UL1TR000050 - Diese Arbeit wurde von der American Heart Association Zuschuss 11SDG5230003 zu GKC und National Center for Advancing Translationale Wissenschaft unterstützt.

1. Zelltransfektion <ol><li> Teller Zellen am Tag vor der Transfektion Plattieren weniger Zellen für Immunfluoreszenz. <ol><li> Für Immunfluoreszenz: in einer 6-Well-Platte, Mantel Glasdeckgläschen (1 pro Well) mit 0,02% Gelatine bei 37 ° C für mindestens 4 Stunden, einmal abwaschen mit Medium und für jeden gut, Platte 1 x 10 5 HEK 293T Zellen in 2 ml komplettem Wachstumsmedium [Dulbecco-modifiziertem Eagle-Medium (DMEM) mit 10% FBS]. </li><li> Für Durchflusszytometrie und Western-Blot-Analysen: Platte 1,2 x 10 5 HEK 293T Zellen / Vertiefung in 2 ml komplettem Wachstumsmedium in 6-Well-Platte (0,3 x 10 5 HEK 293T-Zellen in 0,5 ml komplettem Wachstumsmedium pro Vertiefung in 24-Well-Platte ). </li></ol></li><li> Bereiten DNA für die Transfektion gemäß Herstellerangaben Protokoll: Effectene (Qiagen) zur Immunfluoreszenz eingesetzt. TRANSIT-LT1 (Mirus) ist für durchflusszytometrische und Western-Blot-Analysen verwendet. Anzahl der DNA verwendet wird unten aufgeführt. <table border="1"><tbody><tr><td&gt; </td><td> 24-Well-Platte (ng) </td><td> 6-Well-Platte (ng) </td></tr><tr><td> CFP-Vektor </td><td> 10 </td><td> 50 </td></tr><tr><td> VN N-AKAP-Lbc </td><td> 200 </td><td> 1000 </td></tr><tr><td> VC N-PDE4D3-FL (full-length PDE4D3) </td><td> 2,5, 5, 10, 20, 40, 80 </td><td> 100 </td></tr><tr><td> VC-N PDE4D3-UCR1 (a PDE4D3 N-terminale Fragment mit dem UCR1 Domain) </td><td></td><td> 100 </td></tr><tr><td> VC-N PDE4D3-UCR2 + CAT (a PDE4D3 C-terminalen Fragments mit den UCR2 und katalytischen Domänen) </td><td></td><td> 100 </td></tr></tbody></table> Transfektion unter Verwendung Effectene (6-well): <ol><li> In angegebene Menge von Plasmid-DNA + 4 ul Enhancer zu 100 ul Puffer EC, Mischung und Inkubation für 5 min bei Raumtemperatur. </li><li> In 5 ul Effectene, Mischung und Inkubation für 10 min beiRaumtemperatur. In den Zellen tropfenweise. </li></ol> Transfektion mit Transit-LT1 (6-well/24-well): <ol><li> In angegebene Menge an Plasmid-DNA zu 250 ul μl/50 Opti-MEM I serumfreiem Medium in ein steriles Röhrchen. </li><li> In TRANSIT-LT1 Reagenz zu der verdünnten DNA-Gemisch und Pipette vorsichtig mischen. (Transit-LT1 Reagenz: DNA-Verhältnis ist 3 ul TransIT-LT1 Reagenz pro 1 ug DNA). </li><li> Inkubation für 30 min bei Raumtemperatur und fügen Sie tropfenweise zu den Zellen. </li></ol></li></ol> 2. Handy Vorbereitung für die Durchflusszytometrie, Western Blot und Immunfluoreszenz <ol><li> 24 Stunden nach der Transfektion überprüfen Fluoreszenz unter Epifluoreszenzmikroskop Zellen vor der Ernte. </li><li> Zellpräparat zur durchflusszytometrische Analyse: <ol><li> Waschen der Zellen mit 0,5 ml / 2 ml eiskaltem PBS 24-well/6-well Platten. </li><li> Trennen Zellen mit 50 μl/250 ul 0,05% Trypsin. </li><li> Neutralisieren Trypsin mit 200 ul / 1 ml DMEM-Medium. </li><li> Collect 250 ul Zellen (sowohl für 24-Well-und 6-Well-Platte) durch Zentrifugation bei 500 xg für 5 min, verwenden Sie die restlichen Zellen (1 ml) für Western-Blot-Analyse. </li><li> Die Zellen in 250 ul FACS-Puffer [PBS + 0,5% (w / v) BSA + 2 mM EDTA], auf Eis lagern für durchflusszytometrische Analyse. Die Zellen können auch in 1% Paraformaldehyd fixiert werden (in PBS), wenn Durchflusszytometrie nicht sein wird sofort. </li></ol></li><li> Handy Vorbereitung für Western-Blot-Analyse: parallel durchgeführten mit Zellpräparat zur Durchflusszytometrie. <ol><li> Waschen der Zellen 1x mit 2 ml eiskaltem PBS lysiert Zellen durch Zugabe von 100 ul Lysepuffer [10 mM Natriumphosphat-Puffer, pH 6,95, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1% Triton X-100] . </li><li> Sammeln Zelllysat durch Zentrifugation für 10 min bei 21.000 × g bei 4 ° C. </li><li> Quantifizierung Protein-Konzentration von Bradford-Protein-Assay, laden Sie gleiche Mengen von Protein pro Spur für SDS-PAGE und Transfer auf Nitrozellulose für Immunoblot. </li></ol&gt; </li><li> Zellpräparat zur Immunfluoreszenz. <ol><li> Spülen Zellen 3x mit 2 ml PBS, fixieren Zellen in 1 ml 3,7% Paraformaldehyd (in PBS) für 10 min bei Raumtemperatur, dann waschen mit 2 ml PBS 3x für 5 min. </li><li> Montieren Deckglas auf einem Objektträger aus Glas, mit Montage Medium. Deckglasränder Kanten mit Nagellack, wenn nötig. </li><li> Bewahren Sie die Objektträger bei 4 ° C vor der Prüfung. </li></ol></li></ol> 3. Durchflusszytometrie und Immunfluoreszenz-Mikroskopie <ol><li> Durchflusszytometrie unter Verwendung eines Beckman Coulter Cyan ADP mit 488 nm ausgestattet, 405 nm und 642 nm Festkörperlaser. Summit Software zur Erfassung und Analyse verwendet. <ol><li> Kalibrieren Durchflusszytometer unter Verwendung von Standard-Perlen. </li><li> Grundstück Vorwärtsstreuung (FSC) / Seitwärts Streulicht (SSC) auf einer linearen Skala für Gating Live-Zell-Population. </li><li> Plot FSC / Spannungsimpulsbreite für Gating nicht aggregierten Live-Zell-Population. </li><li> Grundstück count/FL6 (CFP fluorescence, 405 nm) auf einer logarithmischen Skala für Gating nicht aggregierten Live-GFP-positiven Zellen. </li><li> Grundstück count/FL1 (YFP Fluoreszenz, 488 nm) auf einer logarithmischen Skala für BiFC Intensität. </li><li> Führen YFP-CFP-double negative Proben, stellen FSC, SSC, FL1 und FL6 Photomultiplier (PMT) Spannung, um die Schwelle für jedes Signal gesetzt. </li><li> Führen einzigen positiven (beide YFP und CFP + +) Proben für zukünftige off-line Entschädigung </li><li> Erwerben Sie mindestens 10.000 gated Veranstaltungen (nicht aggregierten Live GFP +-Zellen) für die Analyse. </li></ol></li><li> Immunfluoreszenzmikroskopie wird unter Verwendung eines Zeiss LSM 510 konfokalen Mikroskop. Hinweis: es ist wichtig, um alle Einstellungen (wie die Zoom, Lochkamera, Detektor Gain Verstärker-Offset, Baugröße, Scan-Geschwindigkeit, Scan-Durchschnitt, und Laserleistung) konsistent zu halten Proben für angemessenen Vergleich aller Daten. </li></ol> 4. Data Analysis (durchgeführt mit Summit Software) <ol><li> Richten Sie die Analyse-Protokoll ähnlich wie bei acquisition mit der richtigen Gating: Gate 1 in FSC / SSC Dot-Plot für lebende Zellen. Gate 2 in FSC / Pulsbreite von Gate 1 für nicht aggregierte lebenden Zellen. Gate 3 in count / CFP von Gate 3 für nicht-aggregierten GFP + lebenden Zellen. </li><li> Kompensieren YFP und CFP-Signal mit YFP und CFP + + einzigen positiven Zellen. </li><li> Neu zu bestimmen Cut-off-Linien für CFP / YFP-positiven Zellen. </li><li> Export YFP mittlere Fluoreszenzintensität (MFI) von GFP + Zellen in Excel für Daten-Plotten. </li><li> Normalisieren YFP MFI zu Expression von AKAP-Lbc, PDE4D3 und Ladekontrolle α-Tubulin, wie durch Western Blot bestimmt. </li></ol>

<ol>
	<li>Collura, V., Boissy, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+protein-protein+complexes+to+interactomics.">From protein-protein complexes to interactomics.</a> Subcell Biochem. 43, 135-183 (2007).</li><li>Stumpf, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+the+size+of+the+human+interactome.">Estimating the size of the human interactome.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 6959-6964 (2008).</li><li>Shekhawat, S. S., Ghosh, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split-protein+systems:+beyond+binary+protein-protein+interactions.">Split-protein systems: beyond binary protein-protein interactions.</a> Curr. Opin. Chem. Biol. 15, 789-797 (2011).</li><li>Dwane, S., Kiely, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+used+to+study+how+protein+complexes+are+assembled+in+signaling+cascades.">Tools used to study how protein complexes are assembled in signaling cascades.</a> Bioeng. Bugs. 2, 247-259 (2011).</li><li>Piehler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+methodologies+for+measuring+protein+interactions+in+vivo+and+in+vitro.">New methodologies for measuring protein interactions in vivo and in vitro.</a> Curr. Opin. Struct. Biol. 15, 4-14 (2005).</li><li>Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+implementation+of+bimolecular+fluorescence+complementation+(BiFC)+assays+for+the+visualization+of+protein+interactions+in+living+cells.">Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells.</a> Nat. Protoc. 1, 1278-1286 (2006).</li><li>Shyu, Y. J., Suarez, C. D., Hu, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+ternary+complexes+in+living+cells+by+using+a+BiFC-based+FRET+assay.">Visualization of ternary complexes in living cells by using a BiFC-based FRET assay.</a> Nat. Protoc. 3, 1693-1702 (2008).</li><li>Wong, K. A., O'Bryan, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimolecular+fluorescence+complementation.">Bimolecular fluorescence complementation.</a> J. Vis. Exp. (50), e2643 (2011).</li><li>Sugarbaker, E. V., Thornthwaite, J. T., Temple, W. T., Ketcham, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry:+general+principles+and+applications+to+selected+studies+in+tumor+biology.">Flow cytometry: general principles and applications to selected studies in tumor biology.</a> Int. Adv. Surg. Oncol. 2, 125-153 (1979).</li><li>Koshy, S., Alizadeh, P., Timchenko, L. T., Beeton, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+GLUT4+translocation+to+the+plasma+membrane+by+flow+cytometry.">Quantitative measurement of GLUT4 translocation to the plasma membrane by flow cytometry.</a> J. Vis. Exp. (45), e2429 (2010).</li><li>Smith, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+confocal+microscopy.">Basic confocal microscopy.</a> Curr. Protoc. Mol. Biol. Chapter 14 (Unit 14), 11 (2008).</li><li>Westwick, J. K., Lamerdin, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+drug+discovery+with+contextual+assays+and+cellular+systems+analysis.">Improving drug discovery with contextual assays and cellular systems analysis.</a> Methods Mol. Biol. 756, 61-73 (2011).</li><li>Kilpatrick, L. E., Holliday, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+pharmacology+of+G+protein-coupled+receptor+signaling+complexes+using+bimolecular+fluorescence+complementation.">Dissecting the pharmacology of G protein-coupled receptor signaling complexes using bimolecular fluorescence complementation.</a> Methods Mol. Biol. 897, 109-138 (2012).</li></ol>

Die Autoren erklären, dass sie keine finanziellen Interessen haben.

BiFC ist eine einfache und sensitive Methode zur Protein-Protein-Wechselwirkung zu untersuchen. Diese Methode kann verwendet werden, um neue Protein-Protein-Interaktionen zu identifizieren, ist es jedoch besonders günstig, um Protein-Protein-Wechselwirkung innerhalb der Zellen zu bestätigen und funktionellen Eigenschaften zu untersuchen, wie subzelluläre Lokalisation von Proteinkomplexen, die Kartierung von Protein-Protein-Wechselwirkung Sites und für das Screening von kleinen Molekülen / Peptide, die Modulation vo...

650.000 Protein-Protein-Wechselwirkungen werden geschätzt, um in den menschlichen interactome existieren, spielen eine entscheidende Rolle bei der Aufrechterhaltung der normalen Zellfunktionen 1,2. Neben Co-Immunopräzipitation (Co-IP), der Goldstandard Protein-Protein-Interaktion von Zelllysat studieren, mehrere Proteinfragments Komplementierungsassays (PCA) wurden entwickelt, um die Empfindlichkeit bei der Erkennung von Protein-Protein-Interaktion innerhalb der Zellen 3 verbessern. Techniken umfassen Förster-Resonanz-Energie-Transfer (FRET), Biolumineszenz-Resonanz-Energie-Transfer (BRET) und Fluoreszenz-Komplementation Bimolekulare (BiFC) 4,5. , Die jeweils zu einem potenziellen Partner interagierende Protein (in diesem Beispiel verwenden wir AKAP-Lbc und PDE4D3) verschmolzen sind; BiFC auf dem erleichtert Assoziation von zwei Fragmente eines fluoreszierenden Proteins (YFP eine Variante hier benutzen wir Venus) basiert. Wechselwirkung der beiden Proteine ​​von Interesse in den Zellen führt zu Funktionsstörungen Fluoreszenz, die sichtbar gemacht werden kann fluorescence Mikroskopie mit entweder live oder fixierten Zellen 6,7,8. Im Vergleich zu anderen PKA ist BiFC empfindlicher und weniger technisch anspruchsvoll, mit Potenzial, die zelluläre Lokalisation in lebenden Zellen zu studieren. Ein wesentlicher Nachteil dieses Verfahrens ist jedoch, daß einmal gebildet, das fluoreszierende Protein-Komplex kann nicht rückgängig gemacht werden. Daher ist es nicht eine gute Methode, um dynamische Protein-Protein-Wechselwirkung zu untersuchen. In diesem Papier, verwenden wir, um die Interaktion BiFC Websites von PDE4D3 mit AKAP-Lbc Karte durch die Überwachung der Fluoreszenz-Intensitäten der Venus. Im Vergleich zu herkömmlichen Analyse mittels Fluoreszenzmikroskopie, die zeitaufwendig und arbeitsintensiv (falls nicht mit automatisierten Hochdurchsatz-Maschinen durchgeführt) ist, bietet die Durchflusszytometrie eine einfache quantitative Analyse von Tausenden von Zellen in einer heterogenen Population über eine kurze Zeit 9, 10. Hier wird durch die Durchführung einer Side-by-Side-Vergleich der Durchflusszytometrie-Analyse und BiFC traditionellen Ko-IP, zeigen wir, dass die beiden methoden vergleichbare Daten ist jedoch durchflusszytometrische Analyse BiFC weniger zeitaufwendig und benötigt weniger Material, das nützlicher sein kann, wenn die Zellen von Interesse sind begrenzt.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>Dulbecco's modified Eagle's media (DMEM)</td>
			<td>Invitrogen</td>
			<td>11965-092</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicilin-Streptomycin (pen/strep), 100x</td>
			<td>Invitrogen</td>
			<td>15070-063</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Invitrogen</td>
			<td>16000-044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-Flag antibody</td>
			<td>Sigma</td>
			<td>M2, F1804</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-HA antibody</td>
			<td>Covance</td>
			<td>16B12, MMS-101R</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&alpha;-tubulin</td>
			<td>Sigma</td>
			<td>DM1A, T9026</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-GFP antibody</td>
			<td>Clontech</td>
			<td>632569</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell culture plates, 6-well tissue culture treated</td>
			<td>Thermo Fisher Scientific</td>
			<td>130184</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEK 293T cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Maxiprep plasmid purification kit, high speed</td>
			<td>Qiagen</td>
			<td>12663</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbecco's Phosphate-buffered saline (DPBS), sterile 1x</td>
			<td>Invitrogen</td>
			<td>14190-144</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA, 0.05% (w/v)</td>
			<td>Gibco</td>
			<td>25300</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polystyrene round-bottom tubes for FACS staining</td>
			<td>BD Biosciences</td>
			<td>352052</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>S74337MF</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Prolong gold antifade reagent with DAPI</td>
			<td>Invitrogen</td>
			<td>P-36931</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Superfrost plus Microscope slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fisherfinest premium cover glass</td>
			<td>Fisher Scientific</td>
			<td>12-548-5P</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>CO2 air-jacketed Incubator</td>
			<td>NuAIR DH autoflow</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Confocal microscope LSM510 META</td>
			<td>Carl Zeiss, Inc</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electrophoresis and transfer unit</td>
			<td>Biorad</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cyan ADP Flow Cytometer</td>
			<td>Beckman Coulter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

flow cytometric analysis of bimolecular fluorescence complementation: a high throughput quantitative method to study protein-protein interaction

Unter den Verfahren zur Protein-Protein-Wechselwirkungen in Zellen zu studieren, ist Bimolekulare Fluoreszenz Komplementation (BiFC) relativ einfach und empfindlich. BiFC ist auf die Erzeugung von Fluoreszenz mit zwei nicht-fluoreszierenden Fragmente eines fluoreszierenden Proteins (Venus, gelb fluoreszierendes Protein Variante wird hier verwendet werden). Nicht fluoreszierende Venus Fragmente (VN und VC) mit zwei interagierende Proteine ​​(in diesem Fall AKAP-Lbc und PDE4D3) fusioniert, wodurch Fluoreszenz aufgrund VN-AKAP-LBC-VC-PDE4D3 Interaktion und der Bildung eines funktionellen fluoreszierendes Protein innerhalb der Zellen. BiFC liefert Informationen über die subzelluläre Lokalisation von Proteinkomplexen und der Stärke der Protein-Wechselwirkungen auf Fluoreszenzintensität. Allerdings ist BiFC Analyse unter Verwendung der Mikroskopie, die Stärke der Protein-Protein-Interaktion zu quantifizieren zeitaufwendig und etwas subjektiv durch Heterogenität in der Proteinexpression und Interaktion. Durch die Kopplung durchflusszytometrischenAnalyse BiFC Methodik kann die fluoreszierende BiFC Protein-Protein-Wechselwirkung Signal genau für eine große Anzahl von Zellen in einer kurzen Zeit gemessen werden. Hier zeigen wir eine Anwendung dieser Methode auf Regionen in PDE4D3, die für die Interaktion mit AKAP-Lbc erforderlich sind, abzubilden. Dieser hohe Durchsatz-Screening Methode können Faktoren, die Protein-Protein-Interaktion reguliert angewendet werden.

AKAP-Lbc und PDE4D3 Konstrukte (1B) zu VN und VC Fragmente fusioniert sind. Wechselwirkung von AKAP-Lbc und PDE4D3 führt zu Funktionsstörungen YFP-Fluoreszenz (Abb. 1A). Hier verwenden wir die BiFC Verfahren zur Karte AKAP-LBC-Bindungsstellen in PDE4D3. Stromaufwärts konservierten Region 1 (UCR1) stromaufwärts konservierten Region 2 (UCR2) und katalytischen Bereich (CAT) werden unter PDE4-Proteine ​​konserviert daher Länge (FL), UCR1 und UCR2 plus Katzen Zum Screening wurden di...

Watch this Scientific Journal Video about Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction at JoVE.com

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Durchflusszytometrieanalyse bimolekularer Fluoreszenz Komplementation bietet einen hohen Durchsatz quantitative Methode zur Protein-Protein-Wechselwirkung zu untersuchen. Diese Methode kann zur Zuordnung Proteinbindungsstellen und zum Screening von Faktoren, die Protein-Protein-Interaktion reguliert angewendet werden.

Durchflusszytometrieanalyse bimolekularer Fluorescence Komplementierung: A High Throughput Quantitative Methode zur Protein-Protein Interaction Study

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

flow-cytometric-analysis-bimolecular-fluorescence-complementation

Research

JoVE Journal

Biology

18.3K Aufrufe.  University of Illinois at Chicago. Durchflusszytometrieanalyse bimolekularer Fluoreszenz Komplementation bietet einen hohen Durchsatz quantitative Methode zur Protein-Protein-Wechselwirkung zu untersuchen. Diese Methode kann zur Zuordnung Proteinbindungsstellen und zum Screening von Faktoren, die Protein-Protein-Interaktion reguliert angewendet werden.

Serie: Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Eine Neuronale und Astrocyte Co-Kultur-Assay für High Content Analyse der Neurotoxizität

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Forschung

Biologie

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

Ein High-Throughput-Verfahren zur global Studie der Organellen-Morphologie in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

Bimolekularen Fluoreszenz Komplementation (BIFC) Assay für Protein-Protein-Wechselwirkungen in Onion Zellen unter Verwendung des Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Bimolecular Fluorescence Complementation

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1)1,2. A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible3. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions4. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions5,6. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and F&ouml;rster resonance energy transfer (FRET)7. For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments8. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling.

The subcellular localization of proteins is important in determining the spatio-temporal regulation of cell signaling. Here, we describe bimolecular fluorescence complementation (BiFC) as a straightforward method for monitoring the spatial interactions of proteins in the cell.

Bimolekularen Fluoreszenz Komplementation

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

Detektion von Protein-Wechselwirkungen in Plant mit einem Gateway kompatibel bimolekularen Fluoreszenz Komplementation (BIFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

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 ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

Quantitative Analyse von Autophagie mit Advanced 3D Fluoreszenzmikroskopie

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Ein hoher Durchsatz MHC II-Bindungsassay für die quantitative Analyse von Peptid-Epitope

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Protein-Protein-Wechselwirkungen durch Bimolekulare Fluoreszenz-Komplementation in Tabak-Protoplasten-und Blätter-Visualisierte

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

Hochdurchsatz-Analyse von Bild Tumorsphäroide: Eine benutzerfreundliche Software-Anwendung, um die Größe der Sphäroide automatisch und präzise messen

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...