C. Elegans -Stämme, die in dieser Studie verwendeten stammen vom Caenorhabditis Genetik entfernt, die von den National Institutes of Health - Büro der Forschung Infrastrukturprogramme (P40 OD010440) unterstützt wird. Diese Arbeit wurde unterstützt von NIH: 1R03AG048578-01 Jun Liang, CUNY-CCRG 1501 Jun Liang und PSC-CUNY 66184-00 44 und 67248-00 45 bis Jun Liang. Wir danken Cathy Savage-Dunn freundlicherweise ihr Labor Raum teilen. Alle Fluoreszenzbilder wurden am Queens College Core Facility gesammelt. Wir danken William für seine Bemerkungen über das Manuskript.

(1) Lösungen
<ol><li>
NGM Platten
	<ol>
<li>Fügen Sie in einem 2 L-Erlenmeyer-Kolben 3 g NaCl, 17 g Agar, 2,5 g Pepton und 975 mL dH2O. Cover der Mündung des Kolbens mit Alu-Folie. Autoklaven der Küvette für 50 min. Abkühlen für 20-30 min.</li>
		<li>Fügen Sie dann sterilisierte Lösungen: 1 mL 1 M CaCl2, 1 mL 5 mg/mL Cholesterin in Ethanol, 1 mL 1 M MgSO4und 25 mL 1 M KPO4 (pH 6.0) Puffer. Schwenken Sie die Lösungen, die ihnen gut mischen.</li>
		<li>Mit einem liquid Dispenser, verzichten Sie die NGM-Lösung für 60 mm Petri Platten. Die Platten 2/3 voll Füllen des Nährbodens. In einem luftdichten Behälter bei 4 ° C für eine spätere Verwendung zu speichern.</li>
	</ol>
</li>
	<li>
1 M KPO4 Puffer (pH 6.0)
	<ol>
<li>Lösen Sie 10,83 g KH2PO4 und 3,56 g K2HPO4 dH2O auf, dann stellen Sie das Gesamtvolumen von 100 mL. Autoklaven für 15 Minuten die Lösung.</li>
	</ol>
</li>
	<li>
M9
	<ol>
<li>Lösen Sie 3 g KH2PO4, 6 g Na2HPO4und 5 g NaCl in dH2O, fügen Sie 1 mL 1 M MgSO4, und passen Sie das Gesamtvolumen von 1 L.</li>
	</ol>
</li>
	<li>
10 µg/mL DAPI
	<ol>
<li>Fügen Sie 1 µL von 10 mg/mL DAPI in 999 µL dH2O.</li>
	</ol>
</li>
	<li>
95 % Alkohol (V/V)
	<ol>
<li>95 mL 100 % Alkohol zu messen und zum Einstellen der Lautstärke bis 100 mL destilliertes Wasser hinzufügen.</li>
	</ol>
</li>
	<li>
30 % Aceton (V/V)
	<ol>
<li>DH2O 30 mL Aceton hinzu und passen Sie das Gesamtvolumen von 100 mL.</li>
	</ol>
</li>
</ol>2. Hitze-Stress
<ol><li>Generieren einer Extrachromosomale Array-Linie mit einem translationalen Konstrukt Ziel Gene9,10. Alternativ erhalten Sie solche Linien von Caenorhabditis Genetik Center (CGC), die eine öffentliche Ressource für C. Elegans Forschung ist, oder aus einer zuvor veröffentlichten Forschungslabor.</li>
	<li>Die Extrachromosomale Linien in das Genom zu integrieren, Würmer bis 3.800 Rad γ-Strahlung9auszusetzen. Wählen Sie die stabil ausgedrückten Linien so, dass jedes Tier ein Marker Protein wie GFP oder Walze drückt.</li>
	<li>Um während der Strahlung erzeugte Mutationen zu entfernen, outcross Linien mindestens 2 X. Verwenden Sie diese transgenen Linie Protein Ausdruck Muster Änderungen unter Stress zu erkennen.</li>
	<li>Bereiten Sie NGM Wurm Platten vor, wie unter Punkt 1.1 beschrieben. Streifen einen OP50-Klon und Kultur die Bakterien in flüssiger LB Brühe über Nacht. Samen, die der NGM-Platten mit flüssigen OP50 Kultur und die Platten in einem Inkubator 37 ° C inkubieren, über Nacht in eine Bakterien-Rasen als Nahrungsquelle für die Würmer wachsen. Die Platten bei Zimmertemperatur mindestens 30 Minuten vor der Anwendung abkühlen.</li>
	<li>Wachsen der transgenen Linie bei 20 ° C ohne Hunger für mindestens 2 Generationen vor der Hitze-Stress-test.</li>
	<li>Wählen Sie 8 – 10 jungen trächtige Erwachsene (Gebärmutter voller Eier) zu einem neuen Wurm-Platte, dann lassen sie Eier für ca. 3 – 5 h und entfernen Sie die Erwachsenen von den Platten zu.</li>
	<li>Um die Würmer zu synchronisieren, lassen Sie die Eier schlüpfen und wachsen die Larven für ca. 48 h auf dem vierten Larvenstadium (L4). Untersuchen Sie ihre Vulva-Struktur (eine halbmondförmige Struktur) Ermittlung der L4-Stadium (Abb. 1, Pfeil)11.</li>
	<li>Platzieren Sie die Wurm-Platten mit den L4-Larven in einem Inkubator 35 ° C für 2 – 5 h, Hitzestress zu erreichen. Legen Sie einen Thermometer in den Inkubator zur Messung der Temperatur.</li>
</ol>(3) DAPI-Färbung
<ol><li>
Ethanol-Fixierung
	<ol>
<li>Fügen Sie 10 µL des Puffers M9 in der Mitte des einen Objektträger.</li>
		<li>Wähle die Hitze schockiert Würmer aus Schritt 2,8 und lege sie in den M9-Puffer auf dem Objektträger.<ol>
<li>Alternativ waschen Sie die Platte mit M9, 1.000 x g für 1 min bei Raumtemperatur zentrifugiert und den überstand verwerfen. Fügen Sie die Würmer auf den Objektträger.</li>
		</ol>
</li>
		<li>Verwenden Sie ein weiches Gewebe, um überschüssige Flüssigkeit abtropfen lassen. Sehen Sie sich diesen Schritt unter dem sezierenden Mikroskop.</li>
		<li>Fügen Sie 10 µL von 95 % Alkohol auf die Würmer und an der Luft trocknen lassen. Beobachte den Prozess unter dem sezierenden Mikroskop, und lassen Sie sich nicht die Tiere zu trocken zu bekommen. Sofort nachdem das Äthanol von den Tieren verdunstet ist, fahren Sie fort mit dem nächsten Schritt fort. Hinweis: Ethanol verdunstet sehr schnell.</li>
		<li>Wiederholen Sie Schritt 3.1.4 zweimal so dass die Würmer fixiert sind. Hinweis: Es ist normal, dass die Tiere sich dunkler und verzerrt.</li>
		<li>Fügen Sie 10 µL des DAPI (10 µg/mL), die Würmer. Sofort mit einem Deckglas abdecken und die Folie mit transparenten Nagellack Gel zu versiegeln.</li>
		<li>Die Tiere nach etwa 10 min auf dem Fluoreszenzmikroskop ansehen.</li>
	</ol>
</li>
	<li>
Aceton-Fixierung (Alternativ)
	<ol>
<li>Waschen Sie die Hitze schockiert Platte aus Schritt 2.8 mit M9, 1.000 x g für 1 min bei Raumtemperatur Zentrifugieren Sie und verwerfen Sie überstand. Waschen Sie die Tablette mit 1 mL dH2O, sammeln Sie das Pellet und verwerfen Sie den überstand.</li>
		<li>Fügen Sie 400 µL 30 % Aceton für 15 min. Zentrifugieren bei 1.000 X g für 1 min bei Raumtemperatur und verwerfen Sie den überstand. Verwenden Sie 500 µL dH2O Waschen der Tiere 2 X. Verwerfen Sie den überstand.</li>
		<li>Fügen Sie 200 µL DAPI (10 µg/mL) oder 4 x die Menge an der gesamten Würmer Volumen in der Röhre für 15 min.</li>
		<li>Bei 1.000 X g für 1 min zentrifugieren und den überstand verwerfen. Waschen Sie das Pellet 2 X mit 500 µL dH2O. legen die Würmer auf Objektträger, decken Sie sie mit Deckgläsern, die Folie mit transparenten Nagellack Gel zu versiegeln und beobachten die Tiere unter dem Fluoreszenzmikroskop.</li>
	</ol>
</li>
</ol>(4) mikroskopische Bildgebung
<ol><li>Schalten der UV-Lichtquelle und dem Fluoreszenzmikroskop. Schalten Sie den Computer mit dem Mikroskop verbunden.</li>
	<li>Laden Sie die Folie auf dem Fluoreszenzmikroskop. Verwenden Sie eine low-Power-Objektiv (10 X) zu finden die Würmer, erhöhen Sie die Vergrößerung-Kraft auf 400 X und fokussieren Sie auf die Probe.</li>
	<li>Öffnen Sie die Software mit dem Mikroskop zur Verfügung gestellt. Klicken Sie im Menü auf "Übernahme"; Klicken Sie auf "Kamera" und wählen Sie die Kamera an das Mikroskop angeschlossen; Dadurch kann die ausgewählte Kamera, mit der Software zu kommunizieren. Hinweis: Verschiedene Mikroskope variieren in Betrieb.</li>
	<li>Klicken Sie unter den gleichen "Erwerb" auf "Mehrdimensionalen Erfassung". Daraufhin wird eine neue "Multidimensional" Navigation Erfassungsfenster. Klicken Sie auf "Multichannel", und alle verfügbaren Kanäle werden angezeigt.</li>
	<li>Wählen Sie blau, grün und DIC (differential Interferenz-Kontrast), das Präparat zu beobachten. Verlassen Sie im Fenster "Mehrdimensionalen Erfassung" Navigation die "Blau-DAPI", "Green-GLP" und "Grau-DIC", "Nomarski" Kanäle auf; mit der rechten Maustaste auf anderen Kanälen wie "Red-Rhodamin" und "White-helles Feld", um sie zu schließen.</li>
	<li>
Messen Sie zunächst die Belichtungszeit für jeden Kanal:
	<ol>
<li>Klicken Sie auf den "Blau-DAPI" Kanal um es auszuwählen und klicken dann auf "Messen", um ein live-Bild unter dem DAPI-Kanal mit der automatischen Belichtungszeit nehmen. Es erscheint ein neues Fenster mit einem live-Bild.</li>
		<li>Passen Sie auf der linken Seite des Fensters Livebild die Belichtungszeit manuell an, wenn nötig, um das Bild wie gewünscht.</li>
		<li>Klicken Sie auf " ""OK", "in das live-Bild-Fenster. Dadurch wird die Belichtungszeit unter dem DAPI-Kanal.</li>
	</ol>
</li>
	<li>Wiederholen Sie Schritt 4.6 für die anderen Kanäle, "Green-GLP" und "Grau-DIC", "Nomarski", um die Belichtungszeiten für diese Kanäle einrichten.</li>
	<li>Klicken Sie im Navigationsfenster "Mehrdimensionalen Erfassung" auf "Start" um automatisch 3 Bilder unter den 3 verschiedenen Kanälen in Ordnung (siehe oben) mit bestimmten Belichtungszeiten als Set zu sammeln.</li>
	<li>Um die Datei zu speichern, gehen Sie zum Hauptmenü zurück, klicken Sie auf "Datei" dann auf "Speichern unter". Dateinamen und den Ordnernamen eingeben. Speichern Sie die Datei als Standard-Dateiformat, die detaillierten Bildinformationen für die Zukunft zu bewahren.</li>
	<li>
Um die Datei in andere Formate zu exportieren:
	<ol>
<li>Gehen Sie auf "Datei" und klicken Sie auf "Exportieren". Es öffnet sich ein Fenster "Export-Setting".</li>
		<li>Stellen Sie Dateinamen und Ordner. Überprüfen Sie die Daten "Erstellen Sie einen Projektordner" besser zu organisieren. Die Software erlaubt auch dem Benutzer, 4 Weitere Optionen festlegen: "Generate fusionierte Bilder", "Verwenden Sie Farbe für Kanal-Bilder", "Einsatz-Channel-Namen" und "Nur Bild verschmolzen".</li>
		<li>Wählen Sie einen gewünschten Dateityp aus der Drop-Down-Menü, z. B. "TIF", "BMP", "PSD", oder "JPG" und andere Arten. Klicken Sie auf "Start" zu exportieren.</li>
	</ol>
</li>
</ol>

<ol>
	<li>Essers, M. A., 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=FOXO+transcription+factor+activation+by+oxidative+stress+mediated+by+the+small+GTPase+Ral+and+JNK.">FOXO transcription factor activation by oxidative stress mediated by the small GTPase Ral and JNK.</a> The EMBO Journal. 23 (24), 4802-4812 (2004).</li><li>Oh, S. W., 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=JNK+regulates+lifespan+in+Caenorhabditis+elegans+by+modulating+nuclear+translocation+of+forkhead+transcription+factor/DAF-16.">JNK regulates lifespan in Caenorhabditis elegans by modulating nuclear translocation of forkhead transcription factor/DAF-16.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (12), 4494-4499 (2005).</li><li>Gross, A., 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=Caspase+cleaved+BID+targets+mitochondria+and+is+required+for+cytochrome+c+release,+while+BCL-XL+prevents+this+release+but+not+tumor+necrosis+factor-R1/Fas+death.">Caspase cleaved BID targets mitochondria and is required for cytochrome c release, while BCL-XL prevents this release but not tumor necrosis factor-R1/Fas death.</a> The Journal of Biological Chemistry. 274 (2), 1156-1163 (1999).</li><li>Doerflinger, M., Glab, J. A., Puthalakath, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BH3-only+proteins:+a+20-year+stock-take.">BH3-only proteins: a 20-year stock-take.</a> The FEBS Journal. 282 (6), 1006-1016 (2015).</li><li>Mayorova, T. D., 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=Cells+containing+aragonite+crystals+mediate+responses+to+gravity+in+Trichoplax+adhaerens+(Placozoa),+an+animal+lacking+neurons+and+synapses.">Cells containing aragonite crystals mediate responses to gravity in Trichoplax adhaerens (Placozoa), an animal lacking neurons and synapses.</a> PLoS One. 13 (1), e0190905 (2018).</li><li>Terron-Camero, L. C., Molina-Moya, E., Sanz-Fernandez, M., Sandalio, L. M., Romero-Puertas, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+reactive+oxygen+and+nitrogen+species+(ROS/RNS)+during+hypersensitive+cell+death.">Detection of reactive oxygen and nitrogen species (ROS/RNS) during hypersensitive cell death.</a> Methods in Molecular Biology. , 97-105 (2018).</li><li>Pedrazzini, E., Vitale, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+biosynthesis+and+maturation+in+the+ER.">Protein biosynthesis and maturation in the ER.</a> Methods in Molecular Biology. , 179-189 (2018).</li><li>Liang, J., Shaulov, Y., Savage-Dunn, C., Boissinot, S., Hoque, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloride+intracellular+channel+proteins+respond+to+heat+stress+in+Caenorhabditis+elegans.">Chloride intracellular channel proteins respond to heat stress in Caenorhabditis elegans.</a> PLoS One. 12 (9), e0184308 (2017).</li><li>Evans, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+and+microinjection.">Transformation and microinjection.</a> Wormbook. , (2006).</li><li>Mello, C., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+transformation.">DNA transformation.</a> Methods in Cell Biology. 48, 451-482 (1995).</li><li>Yochem, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nomarski+images+for+learning+the+anatomy,+with+tips+for+mosaic+analysis.">Nomarski images for learning the anatomy, with tips for mosaic analysis.</a> Wormbook. , (2006).</li><li>Dulhunty, A., Gage, P., Curtis, S., Chelvanayagam, G., Board, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glutathione+transferase+structural+family+includes+a+nuclear+chloride+channel+and+a+ryanodine+receptor+calcium+release+channel+modulator.">The glutathione transferase structural family includes a nuclear chloride channel and a ryanodine receptor calcium release channel modulator.</a> The Journal of Biological Chemistry. 276 (5), 3319-3323 (2001).</li><li>Suh, K. S., Malik, M., Shukla, A., Yuspa, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLIC4,+skin+homeostasis+and+cutaneous+cancer:+surprising+connections.">CLIC4, skin homeostasis and cutaneous cancer: surprising connections.</a> Molecular Carcinogenesis. 46 (8), 599-604 (2007).</li><li>Yao, Q., Qu, X., Yang, Q., Wei, M., Kong, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLIC4+mediates+TGF-beta1-induced+fibroblast-to-myofibroblast+transdifferentiation+in+ovarian+cancer.">CLIC4 mediates TGF-beta1-induced fibroblast-to-myofibroblast transdifferentiation in ovarian cancer.</a> Oncology Reports. 22 (3), 541-548 (2009).</li><li>Ronnov-Jessen, L., Villadsen, R., Edwards, J. C., Petersen, O. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+a+chloride+intracellular+channel+gene,+CLIC4,+in+transforming+growth+factor-beta1-mediated+conversion+of+fibroblasts+to+myofibroblasts.">Differential expression of a chloride intracellular channel gene, CLIC4, in transforming growth factor-beta1-mediated conversion of fibroblasts to myofibroblasts.</a> The American Journal of Pathology. 161 (2), 471-480 (2002).</li><li>He, G., 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=Role+of+CLIC4+in+the+host+innate+responses+to+bacterial+lipopolysaccharide.">Role of CLIC4 in the host innate responses to bacterial lipopolysaccharide.</a> European Journal of Immunology. 41 (5), 1221-1230 (2011).</li><li>Zhong, J., 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=Inhibition+of+CLIC4+enhances+autophagy+and+triggers+mitochondrial+and+ER+stress-induced+apoptosis+in+human+glioma+U251+cells+under+starvation.">Inhibition of CLIC4 enhances autophagy and triggers mitochondrial and ER stress-induced apoptosis in human glioma U251 cells under starvation.</a> PLoS One. 7 (6), e39378 (2012).</li><li>Liang, J., 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=The+Caenorhabditis+elegans+schnurri+homolog+sma-9+mediates+stage-+and+cell+type-specific+responses+to+DBL-1+BMP-related+signaling.">The Caenorhabditis elegans schnurri homolog sma-9 mediates stage- and cell type-specific responses to DBL-1 BMP-related signaling.</a> Development. 130 (26), 6453-6464 (2003).</li><li>Leopold, L. E., Heestand, B. N., Seong, S., Shtessel, L., Ahmed, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+pairing+during+meiosis+triggers+multigenerational+transgene+silencing+in+Caenorhabditis+elegans.">Lack of pairing during meiosis triggers multigenerational transgene silencing in Caenorhabditis elegans.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (20), E2667-E2676 (2015).</li><li>Schultz, R. D., Bennett, E. E., Ellis, E. A., Gumienny, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+extracellular+matrix+organization+by+BMP+signaling+in+Caenorhabditis+elegans.">Regulation of extracellular matrix organization by BMP signaling in Caenorhabditis elegans.</a> PLoS One. 9 (7), e101929 (2014).</li><li>Matus, D. Q., 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=Invasive+cell+fate+requires+G1+cell-cycle+arrest+and+histone+deacetylase-mediated+changes+in+gene+expression.">Invasive cell fate requires G1 cell-cycle arrest and histone deacetylase-mediated changes in gene expression.</a> Developmental Cell. 35 (2), 162-174 (2015).</li><li>Reddy, K. C., Villeneuve, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+HIM-17+links+chromatin+modification+and+competence+for+initiation+of+meiotic+recombination.">C. elegans HIM-17 links chromatin modification and competence for initiation of meiotic recombination.</a> Cell. 118 (4), 439-452 (2004).</li><li>Ames, K., 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=A+non-cell-autonomous+role+of+BEC-1/BECN1/Beclin1+in+coordinating+cell-cycle+progression+and+stem+cell+proliferation+during+germline+development.">A non-cell-autonomous role of BEC-1/BECN1/Beclin1 in coordinating cell-cycle progression and stem cell proliferation during germline development.</a> Current Biology. 27 (6), 905-913 (2017).</li><li>Pekar, O., 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=Linking+the+environment,+DAF-7/TGFbeta+signaling+and+LAG-2/DSL+ligand+expression+in+the+germline+stem+cell+niche.">Linking the environment, DAF-7/TGFbeta signaling and LAG-2/DSL ligand expression in the germline stem cell niche.</a> Development. 144 (16), 2896-2906 (2017).</li><li>Schvarzstein, M., 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=DNA+helicase+HIM-6/BLM+both+promotes+MutSgamma-dependent+crossovers+and+antagonizes+MutSgamma-independent+interhomolog+associations+during+caenorhabditis+elegans+meiosis.">DNA helicase HIM-6/BLM both promotes MutSgamma-dependent crossovers and antagonizes MutSgamma-independent interhomolog associations during caenorhabditis elegans meiosis.</a> Genetics. 198 (1), 193-207 (2014).</li><li>Flemming, A. J., Shen, Z. Z., Cunha, A., Emmons, S. W., Leroi, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+polyploidization+and+cellular+proliferation+drive+body+size+evolution+in+nematodes.">Somatic polyploidization and cellular proliferation drive body size evolution in nematodes.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (10), 5285-5290 (2000).</li><li>Nagamatsu, Y., Ohshima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+for+the+control+of+body+size+by+a+G-kinase+and+a+downstream+TGFbeta+signal+pathway+in+Caenorhabditis+elegans.">Mechanisms for the control of body size by a G-kinase and a downstream TGFbeta signal pathway in Caenorhabditis elegans.</a> Genes to Cells. 9 (1), 39-47 (2004).</li></ol>

Die Autoren haben nichts zu verzollen.

Dieser Artikel präsentiert eine schnelle und effiziente Methode, um Protein subzelluläre Änderungen vom Zytoplasma in den Zellkern zu überprüfen. Die Proteinexpression zeigte eine GFP Fusion, während die Kern-Struktur vom DAPI-Färbung (Abbildung 3) überprüft wurde. Da Immunostaining C. Elegans Proteine ist eine Herausforderung, die meisten C. Elegans Protein subzelluläre Lokalisierung zeichnen sich durch Markieren mit markerproteine wie GFP, Laz und mCherry<sup cl...

Eine Änderung der subzellulären Lokalisierung Protein ist ein gemeinsamer Mechanismus in Reaktion auf interne oder externe Signale wie Hitze-Stress, Hunger, oxidativen Stress, Apoptose, Protein-Phosphorylierung und andere. Hitze-Stress induziert z. B. Mitglied FOXO DAF-16 nukleare Translokation1,2und dem Pro-apoptotische BCL-2-Protein, das Gebot zu den Mitochondrien beim empfangenden Tod Signalisierung3,4translocates. Verschiedene Techniken zur Verfügung, diese Veränderungen zu erkennen. Eine Kombination von westliche Beflecken und biochemisch isolieren subzelluläre Strukturen (z.B., Mitochondrien oder die Kerne) könnten auch die Ziel-3erreichen. Es erfordert jedoch einen spezifischen Antikörper gegen das Protein des Interesses. Auf diese Weise wird ein etablierter Antikörper der Schlüssel zum Erfolg. Ein alternativer Ansatz besteht darin, beschriften unterschiedliche subzelluläre Strukturen oder Organellen mit verschiedenen Markern wie grüne Fluoreszenz-Protein (GFP), rote Fluoreszenz-Protein (RFP), gelbe Fluoreszenz-Protein (YFP) und mCherry, und inzwischen Beschriften des Proteins Interesse mit anderen Markern. Dann beobachten sie unter einem confocal Mikroskop, die Ziele5,6zu lokalisieren. Radioaktive Isotope sind eine Alternative für die Kennzeichnung Zielproteine und dann ihre subzelluläre Lokalisation7erkennen. Diese Methode erfordert jedoch angemessene Ausbildung und Behandlung radioaktiver Abfälle. Ein alternativer Ansatz muss unter Umständen wie das Fehlen eines spezifischen Antikörpers, das Fehlen eines richtigen Marker oder die Knappheit von Geräten wie einem confocal Mikroskop betrachtet werden. Um ein Protein nuklearen Translokation zu identifizieren, ist es attraktiv, nur die Zielproteine mit einem Filzstift beschriften und, Kerne mit dem chemischen Reagenz 4', 6-Diamidino-2-Phenylindole (DAPI) da dies nur eine regelmäßige Fluoreszenzmikroskop erfordert zu beflecken.
Immunolabeling C. Elegans mit Antikörpern ist schwierig wegen der geringen Durchlässigkeit der Eierschale oder die kollagenen Nagelhaut rund um das Tier. Unterdessen da C. Elegans Proteine deutlich abweichend von ihrer vertebrate Orthologe sind, bieten einige kommerzielle Firmen C. Elegans mit speziellen Produkten. Es ist schwierig für ein kleines Labor, C. Elegans Antikörper selbst zu generieren. Forscher aus der Gemeinschaft verwenden oft tagged Proteine als Marker, um ein Protein-Lokalisierung oder gen-Expression zu demonstrieren. Dieser Artikel verwendet EXL-1::GFP als Beispiel, um eine Protein nuklearen Translokation unter Hitze-Stress-8zu verfolgen. Eine integrierte translationale Exl-1::gfp in das tierische Genom wird verwendet, um stabil das Gen mit der Gfp Fusion zum Ausdruck bringen. Forschung hat gezeigt, dass Exl-1 in Darm, Körperwand Muskel- und anderen subzellulären Strukturen8ausgedrückt wird. Dieses Protokoll zeigt, wie Würmer, die vierten Larvenstadium (L4) zu synchronisieren, führen Sie eine Hitze Stress Experiment und Verhalten DAPI Färbung, Ethanol Fixierung, Aceton Fixierung und Bildgebung unter dem regulären Fluoreszenzmikroskop.

<table>
	<tbody>
		<tr>
			<td>DAPI</td>
			<td>Biotium</td>
			<td>40043</td>
			<td>both Hoechst 33258 and Hoechst 33342 also works well</td>
		</tr>
		<tr>
			<td>OP50</td>
			<td>CGC</td>
			<td>OP50</td>
			<td>https://cgc.umn.edu/</td>
		</tr>
		<tr>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td></td>
			<td></td>
			<td>https://cgc.umn.edu/</td>
		</tr>
		<tr>
			<td>Kim Wipe</td>
			<td>Kimberly-Clark Corporation</td>
			<td></td>
			<td>soft tissue</td>
		</tr>
		<tr>
			<td>Zeiss&#160; AX10</td>
			<td>Zeiss</td>
			<td></td>
			<td>fluorescence microscope</td>
		</tr>
		<tr>
			<td>Axiovision Rel 4.8</td>
			<td>Zeiss</td>
			<td></td>
			<td>microscope software</td>
		</tr>
		<tr>
			<td>AxioCam MR Rev3</td>
			<td>Zeiss</td>
			<td></td>
			<td>digital camera</td>
		</tr>
		<tr>
			<td>Incubator&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transparent nail polish gel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm petri dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-20 &#181;L pipettor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20-200 &#181;L pipettor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>200-1000 &#181;L pipettor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-200 &#181;L pipet tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>200-1000 &#181;L pipet tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum wire worm pick</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

detecting protein subcellular localization by green fluorescence protein tagging and 4',6-diamidino-2-phenylindole staining in caenorhabditis elegans

In diesem Protokoll, eine grüne Fluoreszenz-Protein (GFP)-Fusionsprotein und 4', 6-Diamidino-2-Phenylindole (DAPI) Färbung dienen zum Nachverfolgen von Protein subzelluläre Lokalisation Änderungen; insbesondere betonen eine nukleare Translokation unter einer Hitze Zustand. Proteine reagieren entsprechend zu externen und internen Signale. Ein gemeinsamer Mechanismus soll seine subzelluläre Lokalisation zu ändern. Dieser Artikel beschreibt ein Protokoll, um Protein-Lokalisierung zu verfolgen, die nicht erfordert einen Antikörper, radioaktive Markierung oder ein confocal Mikroskop. In diesem Artikel wird GFP verwendet, um das Zielprotein EXL-1 in C. Elegans, Mitglied der Chlorid intrazellulären Kanal Proteine (CLICs) Familie, einschließlich Säugetieren CLIC4 tag. Eine integrierte translationale Exl-1::gfp transgene Linie (mit einem Promotor und eine vollständige Gensequenz) entstand durch Umwandlung und γ-Strahlung, und stabil bekundet die gen und GLP. Neuere Forschungen zeigten, dass bei Hitzestress, nicht oxidativen Stress, EXL-1::GFP sammelt sich im Zellkern. Überlappung der GFP-Signal mit der Kerne-Struktur und das DAPI Signale bestätigt der EXL-1 subzelluläre Lokalisation Änderungen unter Stress. Dieses Protokoll stellt zwei unterschiedliche Fixierung Methoden für DAPI-Färbung: Ethanol Fixierung und Aceton Fixierung. Das DAPI-Färbung-Protokoll in diesem Artikel vorgestellten ist schnell und effizient und bewahrt das GFP-Signal und das Protein subzelluläre Lokalisation Änderungen. Diese Methode erfordert nur ein Fluoreszenzmikroskop mit Nomarski, FITC Filter und einem DAPI-Filter. Es eignet sich für kleine Labor, studentische Forschung, High School Student Forschung und Biotechnologie Klassenräume.

Chlorid intrazellulären kanalproteinen (CLIC) sind multifunktionale Proteine, die hoch über Arten12konserviert sind. Ein Großteil der Forschung zeigt, dass CLICs zu, zellulären Stresses, Autophagie, Apoptose, Karzinogenese, Angiogenese und der angeborenen Immunantwort Makrophagen in der Säugetier-System13,14,15,16 regulieren ,<...

Watch this Scientific Journal Video about Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans at JoVE.

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

Dieses Protokoll wird veranschaulicht, wie eine Protein nuklearen Translokation unter Hitzestress nachverfolgen mithilfe eine grüne Fluoreszenz-Protein (GFP)-Fusionsprotein als Marker und 4', 6-Diamidino-2-Phenylindole (DAPI) Färbung. Das DAPI Färbeprotokoll rückt und bewahrt die GFP und Protein subzelluläre Lokalisation Signale.

Erkennung von Protein subzelluläre Lokalisation durch grüne Fluoreszenz Protein Tagging und 4', 6-Diamidino-2-Phenylindole Färbung in Caenorhabditis elegans

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein ...

detecting-protein-subcellular-localization-green-fluorescence-protein

Research

JoVE Journal

Biology

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

9.5K Aufrufe.  Borough of Manhattan Community College/CUNY. Dieses Protokoll wird veranschaulicht, wie eine Protein nuklearen Translokation unter Hitzestress nachverfolgen mithilfe eine grüne Fluoreszenz-Protein (GFP)-Fusionsprotein als Marker und 4', 6-Diamidino-2-Phenylindole (DAPI) Färbung. Das DAPI Färbeprotokoll rückt und bewahrt die GFP und Protein subzelluläre Lokalisation Signale.

Serie: Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

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

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

Forschung

Biologie

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Mit dem GELFREE 8100 Fraktionierung System für Molecular Weight-Based Fraktionierung mit Liquid Phase Wiederherstellung

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Grundlegende<em&gt; Caenorhabditis elegans</em&gt; Methoden: Synchronisation und Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the Mgm101 protein is involved in the recombinational repair of mitochondrial DNA. Recent investigations have indicated that Mgm101 is related to the Rad52-type recombination protein family. These proteins form large oligomeric rings and promote the annealing of homologous single stranded DNA molecules. However, the characterization of Mgm101 has been hindered by the difficulty in producing the recombinant protein. Here, a reliable procedure for the preparation of recombinant Mgm101 is described. Maltose Binding Protein (MBP)-tagged Mgm101 is first expressed in Escherichia coli. The fusion protein is initially purified by amylose affinity chromatography. After being released by proteolytic cleavage, Mgm101 is separated from MBP by cationic exchange chromatography. Monodispersed Mgm101 is then obtained by size exclusion chromatography. A yield of ~0.87 mg of Mgm101 per liter of bacterial culture can be routinely obtained. The recombinant Mgm101 has minimal contamination of DNA. The prepared samples are successfully used for biochemical, structural and single particle image analyses of Mgm101. This protocol may also be used for the preparation of other large oligomeric DNA-binding proteins that may be misfolded and toxic to bacterial cells.

The yeast mitochondrial nucleoid protein, Mgm101, is a Rad52-type recombination protein that forms large oligomeric rings. A protocol is described to prepare soluble recombinant Mgm101 using the Maltose Binding Protein (MBP)-tagging strategy coupled with cation exchange and size exclusion chromatography.

Vorbereitung des Mgm101 Rekombinationsprotein von MBP-based Tagging-Strategie

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

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 ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Green Fluorescent Protein-basierte Screening Expression von Membranproteinen in<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

Die Quantifizierung der Proteinexpression und Co-Lokalisation Mit Multiplexed immunhistochemischen Anfärbung und Multispektraltechnik

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, medial-Golgi, trans-Golgi and trans-Golgi network. Cellular functions of the Golgi are determined by the characteristic distribution of its resident proteins. The spatial resolution of conventional light microscopy is too low to resolve sub-Golgi structure or cisternae. Thus, the immuno-gold electron microscopy is a method of choice to localize a protein at the sub-Golgi level. However, the technique and instrument are beyond the capability of most cell biology labs. We describe here our recently developed super-resolution method called Golgi protein localization by imaging centers of mass (GLIM) to systematically and quantitatively localize a Golgi protein. GLIM is based on standard fluorescence labeling protocols and conventional wide-field or confocal microscopes. It involves the calibration of chromatic-shift aberration of the microscopic system, the image acquisition and the post-acquisition analysis. The sub-Golgi localization of a test protein is quantitatively expressed as the localization quotient. There are four main advantages of GLIM; it is rapid, based on conventional methods and tools, the localization result is quantitative, and it affords ~ 30 nm practical resolution along the Golgi axis. Here we describe the detailed protocol of GLIM to localize a test Golgi protein.

The precise localization of Golgi residents is essential for understanding the cellular functions of the Golgi. However, conventional optical microscopy is unable to resolve the sub-Golgi structure. Here we describe the protocol for a conventional microscopy based super-resolution method to quantitatively determine the sub-Golgi localization of a protein.

Quantitative Lokalisierung eines Golgi-Proteins von Imaging Center der Fluoreszenz Masse

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

Methoden zur Untersuchung Veränderungen inhärenten Proteinaggregation mit dem Alter in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Erkennung von Protein subzelluläre Lokalisation durch grüne Fluoreszenz Protein Tagging und 4', 6-Diamidino-2-Phenylindole Färbung in Caenorhabditis elegans

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

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

Mit dem GELFREE 8100 Fraktionierung System für Molecular Weight-Based Fraktionierung mit Liquid Phase Wiederherstellung

Grundlegende

Vorbereitung des Mgm101 Rekombinationsprotein von MBP-based Tagging-Strategie

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

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

Green Fluorescent Protein-basierte Screening Expression von Membranproteinen in

Die Quantifizierung der Proteinexpression und Co-Lokalisation Mit Multiplexed immunhistochemischen Anfärbung und Multispektraltechnik

Quantitative Lokalisierung eines Golgi-Proteins von Imaging Center der Fluoreszenz Masse

Methoden zur Untersuchung Veränderungen inhärenten Proteinaggregation mit dem Alter in Caenorhabditis elegans