Wir möchten die Lee-Labor der Universität von Minnesota für die Bereitstellung der Maus Rückenmark Gewebeproben, die verwendet wurden bedanken. Diese Arbeit wurde durch das Department of Defense Ovarian Cancer Research Program (OCRP) OC093424 auf MB, die von den Randy Shaver Cancer Research und Gemeinschaftsfonds zur MB und die Minnesota Ovarian Cancer Alliance (MOCA) bis MB. Die Geldgeber hatten keine Rolle in Studiendesign, Datenerhebung und -analyse, Entscheidung, zu veröffentlichen oder zu Erstellung des Manuskripts.

1. Lysis Buffer Vorbereitung <ol><li> Löse Saccharose in deionisiertem (DI) Wasser, um eine 2 M Stammlösung zu machen. Filtern einer 2 M Lösung von Saccharose mit einer Vakuum angetrieben 0,22 um aus Polyvinylidenfluorid (PVDF) Filter. </li><li> Löse Dithiothreitol (DTT) in DI-Wasser, um eine 500 mM-Stammlösung und speichern unter anaeroben Bedingungen zu machen. Löse Magnesiumchlorid (MgCl 2) in DI-Wasser, um eine 100 mM-Stammlösung zu machen. Löse Adenosintriphosphat (ATP) Dinatrium-Hydrat in DI-Wasser, um eine 50 mM Stammlösung zu machen. </li><li> Bilden eine Tris-Lösung bei pH 7,4 durch Auflösen Trizma-Hydrochlorid in entionisiertem Wasser und Einstellen des pH unter Verwendung von Natriumhydroxid (NaOH). </li><li> Kombinieren Abschnitte der Stammlösungen in VE-Wasser auf einen Lysepuffer mit einer Saccharose-Konzentration von 250 mM, DTT einer Konzentration von 1 mM, einer MgCl 2 -Konzentration von 5 mM, einem ATP-Konzentration von 1 mM und einer Tris-Konzentration von 50 mM zu machen . Beispielsweise mischen 535,3 ul Tris, 500 &amp; mgr; l MgCl 2, 1,25 ml Saccharose, 20 &amp; mgr; l DTT, 200 &amp; mgr; l ATP und 7,4947 ml DI-Wasser, um 10 ml Lysepuffer. Dies kann etwa 20 Experimente eingesetzt werden. </li></ol> 2. Kultivieren von Zellen für das Experiment <ol><li> Stellen Medien durch Aliquotieren von 30 ml DMEM + 10% fötalem Rinderserum (FBS) in einem 50 ml konischen Röhrchen. </li><li> Übertragen gefrorenen M17 oder HeLa-Zellen aus Flüssigstickstofflager um einen kleinen Becher mit lauwarmem Wasser. </li><li> Übertragen der Zellen an die 50 ml konischen Röhrchen. Fügen Sie zunächst einige Medien zur Zellfläschchen. Dann übertragen Sie die resuspendierten Zellen innerhalb von 5 Sekunden nach dem Auftauen. </li><li> Zentrifugieren Sie die Zellsuspension bei 450 xg für 5 min, um ein Zellpellet zu erhalten. 20 ml Medium in einem T75-Kolben. </li><li> Entfernen Sie die Medien aus den Zellen mit Absaugung. Mit 5 ml frischem Medium zu dem Zellpellet und resuspendieren. </li><li> Übertragen die 5 ml Zellsuspension in 20 ml Medium in T75-Kolben und Inkubieren bei 37 ° C. Change die Medien alle 3 Tage. </li></ol> 3. Zellernte <ol><li> Entfernen Sie die Medien über Saug-, man aufpassen, die Zellen nicht zu berühren. </li><li> Mit 5 ml phosphatgepufferter Salzlösung (PBS) gewaschen, die Zellen. Entfernen PBS mit Absaugung. </li><li> 3 ml Trypsin-Ethylendiamintetraessigsäure (EDTA) an den T75-Kolben und Inkubieren bei 37 ° C für 3 min. </li><li> 6 ml frischem Medium in den Kolben gegeben und die Zellen resuspendieren. </li><li> Übertragen Sie die Suspension auf eine 15 ml konischen Rohr und Zentrifuge bei 290 xg 5 Minuten. </li><li> Entfernen Sie den Überstand und 5 ml PBS. Klopfen Sie leicht den Boden der Röhre zum Aufbrechen der Pellets. Zentrifuge bei 290 × g für 5 min. </li><li> Wiederholen Sie Schritt 3.6 dreimal. </li><li> Entfernen PBS und Resuspendieren der Zellen in 1 ml frischem PBS. Übertragen auf die Eppendorf-Röhrchen (1,5 ml). Spin bei 290 xg für 5 min. </li></ol> 4. Cell Lysis <ol><li> Messen Sie den ungefähren Volumen des Pellets mit einer Pipette eind abwiegen Glasperlen in einer Masse: Volumen-Verhältnis von 1: 1. Ist die doppelte Volumen des Pellets in Lyse-Puffer. Hinweis: Fügen Sie den Puffer vor der Zugabe der Glasperlen. </li><li> Lysieren die Zellen im Eppendorf-Röhrchen bei der maximalen Rühren für 30 min bei 4 ° C durch Verwirbeln in einem kalten Raum. </li><li> Kurz zentrifugieren bei 200 · g für 30 Sekunden, um die Perlen zu begleichen. Sammeln Sie den Überstand. </li><li> Das gleiche Volumen von Glasperlen als vor dem Überstand. </li><li> Wirbel wieder bei maximaler Rühren für 30 min bei 4 ° C. </li><li> Kurz zentrifugieren bei 200 · g für 30 Sekunden, um die Glasperlen zu begleichen. Sammeln Sie den Überstand. </li><li> Zentrifuge bei 5.030 xg für 5 Minuten, um die Kerne, Membranen und aufgebrochene Zellen zu entfernen. Sammeln Sie den Überstand, der das Zell-Lysat ist. </li></ol> 5. Gewebehomogenisierung <ol><li> Einen Masse: Volumen von 1: 9 Lösung von Maus-Rückenmarkgewebe zu Lysepuffer. Hinweis: Dieses Verfahren ist für eine Vielzahl von unterschiedlichent Gewebeproben. </li><li> Homogenisierung mittels einer Einstellung der Ebene 2 auf der Homogenisator 30 sec lang in das Gewebe, um sicherzustellen, daß es keine Klumpen bleiben. </li><li> Spin down bei 5.030 × g für 3 min, um die Kerne, Membranen und aufgebrochene Zellen zu entfernen. </li><li> Sammeln Sie den Überstand, der das Lysat. </li></ol> 6. Proben Derivatisierung <ol><li> Führen eine Bicinchoninsäure (BCA) in einer 96-Well-Platte Analyse, um die Proteinkonzentration der Gewebelysat Verwendung eines kolorimetrischen 96-Well Plattenlesegerät, wie durch den Pierce BCA Protein Assay Kit Protokoll spezifiziert zu bestimmen. </li><li> Bringen eines Aliquots, die 20 &amp; mgr; g Gesamtprotein zu 50 &amp; mgr; l mit deionisiertem (DI) Wasser. </li><li> Add 2 ul 1,35 uM HA-Ub-Vinylsulfon (VS) zu der Lösung und Inkubation für 1 h bei 37 ° C. Dies führt zu 20 ug Lysat bis 50 nM Sonde in der endgültigen Reaktionsmischung. Dies ist zum großen molaren Überschuß an Kennzeichnungs funktioneller DUBs gewährleisten. </li><li> ICHncubate die Probe in Laemmli-Probenpuffer für 5 Minuten bei 95 ° C. Für eine 52 &amp; mgr; l Reaktionsvolumen Einsatz 26 ul 2x Probenpuffer, 13 ul Probenpuffer 4x oder 6x 8,87 ul Probenpuffer. </li><li> Kühle auf Eis vor dem Laden des Gels. </li></ol> 7. Western Blot <ol><li> Laden Sie eine 12-Well-4-20% Tris-Glycin-Gel mit 40 ul der vorbereiteten Probe </li><li> Führen Sie das Gel bei 95 V, bis der Ionenfront den Boden erreicht. </li><li> Führen Sie eine Übernachtung Transfer des Gels auf eine Polyvinylidendifluorid (PVDF) Membran. </li><li> Inkubieren der Membran in dem Amidoschwarz Fleck und tasten den membane um ein Bild, das die Menge an Protein in jeder Spur erhalten. </li><li> Entfärben der Membran und Inkubation in 5% Milch in PBS für 1 Stunde zu blockieren. </li><li> Inkubieren der Membran in dem primären anti-HA-Antikörper in einer Konzentration von 1: 10.000 in 5% Milch in PBS entweder über Nacht bei 4 ° C oder für 4 h bei Raumtemperatur. </li><li> Führen Sie 3 Waschungen foder jeweils 5 min in PBS mit 0,1% Tween-20 Detergens. </li><li> Inkubieren der Membran in eine Maus Meerrettichperoxidase in einer Konzentration von 1: 10.000 in 5% Milch in PBS für mindestens 2 Std. </li><li> Zuführen 3 Waschungen jeweils 5 min in PBS mit 0,1% Tween-20 Detergens. </li><li> Führen Sie 1 Wasch für 5 min in PBS. </li><li> Inkubieren der Membran in eine chemilumineszierende Nachweisreagenz für 10 min und zu detektieren unter Verwendung eines Chemilumineszenz-Detektor. Verwenden Sie die automatische Belichtungseinstellung auf dem Detektor. </li></ol>

<ol>
	<li>Ovaa, H., Kessler, B. M., Rol&#233;n, U., Galardy, P. J., Ploegh, H. L., Masucci, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-+based+ubiquitin-specific+(USP)+profiling+of+virus-infected+and+malignant+human+cells.">Activity- based ubiquitin-specific (USP) profiling of virus-infected and malignant human cells.</a> Proc Natl Acad Sci USA. 101 (8), 2253-2258 (2004).</li><li>Wilkinson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitination+and+deubiquitination:+Targeting+of+proteins+for+degradation+by+the+proteasome.">Ubiquitination and deubiquitination: Targeting of proteins for degradation by the proteasome.</a> Semin Cell Dev Biol. 11 (3), 141-148 (2000).</li><li>Ziad, M. E., Wilkinson, K. D. <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+proteolysis+by+human+deubiquitinating+enzymes.">Regulation of proteolysis by human deubiquitinating enzymes.</a> Biochim Biophys Acta. 1843 (1), 114-128 (2014).</li><li>Rol&#233;n, U., 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=Activity+Profiling+of+Deubiquitinating+Enzymes+in+Cervical+Carcinoma+Biopsies+and+Cell+Lines.">Activity Profiling of Deubiquitinating Enzymes in Cervical Carcinoma Biopsies and Cell Lines.</a> Mol Carcinog. 45 (4), 260-269 (2006).</li><li>Borodovsky, 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=Chemistry-Based+Functional+Proteomics+Reveals+Novel+Members+of+the+Deubiquitinating+Enzyme+Family.">Chemistry-Based Functional Proteomics Reveals Novel Members of the Deubiquitinating Enzyme Family.</a> Chem Biol. 9 (10), 1149-1159 (2002).</li><li>Andersson, F. L., 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+effect+of+Parkinson&#8217;s-disease-associated+mutations+on+the+deubiquitinating+enzyme+UCH-L1.">The effect of Parkinson&#8217;s-disease-associated mutations on the deubiquitinating enzyme UCH-L1.</a> J Mol Biol. 407 (2), 261-272 (2011).</li><li>Poon, W. 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=&#946;-Amyloid+(A&#946;)+oligomers+impair+brain-derived+neurotrophic+factor+retrograde+trafficking+by+down-regulating+ubiquitin+C-terminal+hydrolase+UCH-L1.">&#946;-Amyloid (A&#946;) oligomers impair brain-derived neurotrophic factor retrograde trafficking by down-regulating ubiquitin C-terminal hydrolase UCH-L1.</a> J Biol Chem. 288 (23), 16937-16948 (2013).</li><li>Nijman, S. 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=The+deubiquitinating+enzyme+USP1+regulates+the+Fanconi+anemia+pathway.">The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway.</a> Mol Cell. 17 (3), 331-339 (2005).</li><li>Stevenson, L. F., Sparks, A., Allende-Vega, N., Xirodimas, D. P., Lane, D. P., Saville, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+deubiquitinating+enzyme+USP2a+regulates+the+p53+pathway+by+targeting+Mdm2.">The deubiquitinating enzyme USP2a regulates the p53 pathway by targeting Mdm2.</a> EMBO J. 26 (4), 976-986 (2007).</li><li>Coughlin, 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=Small-molecule+RA-9+inhibits+proteasome-associated+DUBs+and+ovarian+cancer+in+vitro+and+in+vivo+via+exacerbating+unfolded+protein+responses.">Small-molecule RA-9 inhibits proteasome-associated DUBs and ovarian cancer in vitro and in vivo via exacerbating unfolded protein responses.</a> Clin Cancer Res. 20 (12), 3174-3186 (2014).</li><li>Colla, E., 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=Endoplasmic+Reticulum+Stress+Is+Important+for+the+Manifestations+of+&#945;-Synucleinopathy+In.">Endoplasmic Reticulum Stress Is Important for the Manifestations of &#945;-Synucleinopathy In.</a> Vivo. J Neurosci. 32 (10), 3306-3320 (2012).</li></ol>

Die Autoren haben nichts zu offenbaren.

Seit Ubiquitinierung ist eine grundlegende zelluläre Aktivität, könnte das Verständnis der Regulationsmechanismen der Schlüssel, um auszugraben die Prozesse der Krankheit und Pathologie. Die Verwendung von HA getaggt Ub abgeleitete aktive Zentrum gerichteten Sonden hier berichteten bietet eine einfache, aber sehr zutreffend Methode zur Untersuchung Ub-vermittelten Proteinabbau. Dieses Verfahren ist schneller und weniger kostspielig als das Studium jedes der DUBs einzeln. In diesem Verfa...

Der Ubiquitin-Proteasom-System (UPS) als eine der wichtigsten Abbauwege in der Säugerzelle ist. Substrate für den Abbau im Proteasom gebunden sind kovalent mit Polymeren von Ubiquitin (Ub) 1 markiert. Bevor die gezielte Substrat das Proteasom für den Abbau eintritt, muss der Poly-Ubiquitin-Tag entfernt werden. Eine Klasse von Enzymen als Deubiquitinierungsenzyme (DUBs) bekannt ist, ist für die Beseitigung und Verwertung von Ubiquitin-Moleküle 2 verantwortlich. Es wurde aus dem menschlichen Genom vorhergesagt, dass es fast hundert DUBs arbeiten in der Zelle 3. Mit solch einer großen Anzahl von DUBs Steuerung Ub-vermittelte zelluläre Prozesse, das Studium dieser Enzyme ist eine Herausforderung, da mRNA Techniken nicht geben Auskunft über Aktivität und Western-Blot nur gibt Informationen über die Expressionsniveaus. Die Verwendung von Influenza-Hämagglutinin (HA) versehen, Ub abgeleitete aktive Zentrum gerichteten Sonden ermöglicht eine kovalente modifizierung der funktionellen DUBs und deshalb ergibt eine direkte Visualisierung der Aktivität dieser Enzyme auf einem Western-Blot-4. Die Sonden haben eine C-terminalen Thiol reaktionsfähigen Gruppe, die als Suizid-Substrat für das aktive Zentrum Cysteinrest 5 dient. Bei diesen Sonden ist es möglich, die Aktivität und möglichen Expression vieler DUBs unter beiden pathologischen und physiologischen Zuständen der Zelle zu untersuchen. Änderungen DUB Aktivität wurden in einer Reihe von pathologischen Zuständen, wie Parkinson, Alzheimer, Anämie und verschiedene Krebs 6-10 gebracht. Diese Technik bietet ein leistungsstarkes Werkzeug zur Untersuchung von Krankheiten. In der vorliegenden Arbeit zeigen wir die Anwendung dieser Technik in HeLa und M17-Zellen unter Verwendung von Glasperlen, die aufgeschlossen haben. Zusätzlich beschreiben wir, wie man diese Methode in der Maus Rückenmark Gewebeproben zu verwenden. Die von dieser Technik erhaltenen Informationen können als Ausgangspunkt für die Identifizierung verwendet werdentherapeutische Ziele sowie die Schaffung Modelle für die Untersuchung von verschiedenen Krankheitszuständen. Der wahre Nutzen dieser Technik liegt in ihrer Fähigkeit, Informationen zu mehreren DUBs in einem einzigen Assay bereitzustellen.

<table border="0" cellpadding="0" cellspacing="0" style="width:615px;" width="615">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PowerGen 125 ( Homogenizer)</td>
			<td style="width:113px;">Fischer Scientific</td>
			<td style="width:119px;">14-261-02P</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vacuum-driven filters .22&#181;M&#160;</td>
			<td style="width:113px;"></td>
			<td style="width:119px;">BPV2210</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass beads, acid washed &#8804;106&#181;m (~140 U.S. sieve)</td>
			<td style="width:113px;">Sigma-aldrich</td>
			<td style="width:119px;">G4649-10G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sucrose</td>
			<td style="width:113px;">Fischer Scientific</td>
			<td style="width:119px;">S6-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DL-Dithiothreitol</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:119px;">D0632</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium Chloride</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8266</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Adenosine 5&#39;-triphosphate disodium salt hydrate</td>
			<td style="width:113px;">Sigma-aldrich</td>
			<td style="width:119px;">A26209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trizma hydrochloride&#160;</td>
			<td style="width:113px;">Sigma-aldrich</td>
			<td style="width:119px;">T3253</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:113px;">Life technologies</td>
			<td style="width:119px;">11965-092</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phosphate Buffered Saline</td>
			<td style="width:113px;">Life technologies</td>
			<td style="width:119px;">10010-023</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tissue culture flask 75cm2 w/ filter cup 250 ml 120/ cs</td>
			<td style="width:113px;">Cellstar</td>
			<td style="width:119px;">T-3001-2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">0.05% Trypsin-EDTA (1X)</td>
			<td style="width:113px;">Life technologies</td>
			<td style="width:119px;">25300-054</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HI FBS</td>
			<td style="width:113px;">Life technologies</td>
			<td style="width:119px;">16140-071</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Monoclonal Anti-HA antibody produced in mouse</td>
			<td style="width:113px;">Sigma-aldrich</td>
			<td style="width:119px;">H9658</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ubiquitin vinyl sulfone (HA-tag)</td>
			<td style="width:113px;">Enzo Life Sciences</td>
			<td style="width:119px;">BML-UW0155-0025</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Laemmli&#39;s SDS-Sample Buffer (4X, reducing)</td>
			<td style="width:113px;">Boston BioProducts</td>
			<td style="width:119px;">BP-110R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pierce BCA Protein Assay Kit</td>
			<td style="width:113px;">ThermoScientific</td>
			<td align="right" style="width:119px;">23225</td>
			<td></td>
		</tr>
	</tbody>
</table>

method for measuring the activity of deubiquitinating enzymes in cell lines and tissue samples

Das Ubiquitin-Proteasom-System wurde vor kurzem in verschiedenen Pathologien einschließlich neurodegenerativen Krankheiten und Krebs in Verbindung gebracht. In diesem Licht sind, Verfahren für die Untersuchung der Regulationsmechanismus des Systems wesentlich, die Aufklärung der zellulären und molekularen Prozesse der zuvor genannten Erkrankungen. Die Verwendung von Hämagglutinin abgeleitet Ubiquitin Sonden vorliegenden Ausführungen dient als wertvolles Werkzeug zur Untersuchung des Systems. Dieses Papier Details eines Verfahrens, das dem Benutzer ermöglicht, Tests, die eine direkte Visualisierung der deubiquitinierenden Enzymaktivität geben, durchzuführen. Deubiquitinierungsenzyme steuern proteasomalen Abbau und teilen funktionelle Homologie in ihren aktiven Zentren, die es dem Benutzer, um die Aktivität mehrerer Enzyme in einem Test untersucht werden können. Lysate werden durch sanfte mechanische Zellaufschluss erhalten und mit der aktiven Stelle gerichtet Sonden inkubiert. Functional Enzyme werden mit den Sonden markiert, während inaktiver Enzyme ungebunden bleiben. Durch Laufning dieser Test erhält der Benutzer Informationen sowohl über die Aktivität und möglichen Expression mehrerer Deubiquitinierungsenzyme in einer schnellen und einfachen Weise. Das gegenwärtige Verfahren ist wesentlich effizienter als die Verwendung von einzelnen Antikörper für die vorhergesagten hundert Deubiquitinierungsenzyme in der menschlichen Zelle.

Kultiviert M17 und HeLa-Zellen wurden unter Verwendung des Verfahrens in dem Protokoll (3. Zellernte) detailliert und Maus-Rückenmarksgewebe wurde erhalten geerntet. Das Zellpellet / Rückenmarksgewebe wurde in ein Röhrchen mit der in der Zubereitung der Reagenzien beschrieben Lysepuffer gegeben. Zellpellets mit Glasperlen (1A) und Rückenmark der Maus Gewebeproben wurden unter Verwendung eines Homogenisators (1B) homogenisiert lysiert. Nach der Lyse oder Homogenisierung wurde die Pro...

Watch this Scientific Journal Video about Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples at JoVE.com

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

Auf den aktuellen Protokollinformationen ein Verfahren zur Messung der Aktivität der funktionell homolog Deubiquitinierungsenzyme. Specialized-Sonden, die das Enzym kovalent modifizieren und lassen für die Erkennung. Dieses Verfahren hält das Potenzial, neue therapeutische Targets zu identifizieren.

Verfahren zur Messung der Aktivität von Deubiquitinierungsenzyme in Zelllinien und Gewebeproben

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, ...

method-for-measuring-activity-deubiquitinating-enzymes-cell-lines

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JoVE Journal

Biology

9.5K Aufrufe.  University of Minnesota.  Auf den aktuellen Protokollinformationen ein Verfahren zur Messung der Aktivität der funktionell homolog Deubiquitinierungsenzyme. Specialized-Sonden, die das Enzym kovalent modifizieren und lassen für die Erkennung. Dieses Verfahren hält das Potenzial, neue therapeutische Targets zu identifizieren. 

Serie: Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

Aktin Co-Sedimentation Assay; für die Analyse von Protein-Bindung an F-Aktin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Forschung

Biologie

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &amp;#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

Die Quantifizierung der γH2AX Foci in Gewebeproben

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

Ein Assay zur Messung der Aktivität von Escherichia coli Inducible Lysin Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

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

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

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

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

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

Measuring In Vitro ATPase Activity for Enzymatic Characterization

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization. Proteins hydrolyze ATP in a reaction that results in inorganic phosphate release, and the amount of phosphate liberated is then quantitated using a colorimetric assay. This highly adaptable protocol can be adjusted to measure ATPase activity in kinetic or endpoint assays. A representative protocol is provided here based on the activity and requirements of EpsE, the AAA+ ATPase involved in Type II Secretion in the bacterium Vibrio cholerae. The amount of purified protein needed to measure activity, length of the assay and the timing and number of sampling intervals, buffer and salt composition, temperature, co-factors, stimulants (if any), etc. may vary from those described here, and thus some optimization may be necessary. This protocol provides a basic framework for characterizing ATPases and can be performed quickly and easily adjusted as necessary.

Measuring In Vitro ATPase Activity for Enzymatic Characterization

We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.

Messung<em&gt; In Vitro</em&gt; ATPase Aktivität für die enzymatische Charakterisierung

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate ...

Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility with high-throughput formatting. This protocol describes the use of three different methods for measuring cell proliferation in vitro including conventional hemocytometer counting chamber, a luminescence-based assay that utilizes the change in the metabolic activity of viable cells as a measure of the relative number of cells, and a multi-mode cell imager that measures cell number using a counting algorithm. Each method presents its own advantages and disadvantages for the measurement of cell proliferation, including time, cost and high-throughput compatibility. This protocol demonstrates that each method could accurately measure cell proliferation over time, and was sensitive to detect growth at differing cellular densities. Additionally, measurement of cell proliferation using a cell imager was able to provide further information such as morphology, confluence and allowed for a continual monitoring of cell proliferation over time. In conclusion, each method is capable of measuring cell proliferation, but the chosen method is user-dependent.

This protocol describes the use of three different methods for analyzing cell proliferation in breast cancer cell lines. This includes the use of conventional cell counting, luminescence-based cell viability, and cell counting through the use of a cell imager. Each offers advantages for the reproducible measurement of cell proliferation.

Vergleich von drei verschiedenen Methoden zur Bestimmung Zellproliferation in Brustkrebs-Zelllinien

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility ...

Measuring Lactase Enzymatic Activity in the Teaching Lab

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 &#176;C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

Messung der Laktase enzymatische Aktivität in das Lehrlabor

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Hybrid-Cut: Ein verbesserter Schnittverfahren für widerspenstige Pflanzengewebeproben

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called the auditory bulla houses the ossicles, whereas the auditory capsule encloses the inner ear, namely the cochlea and semicircular canals. Murine ossicles are essential for hearing and thus of great interest to researchers in the field of otolaryngology, but their metabolism, development, and evolution are highly relevant to other fields. Altered bone metabolism can affect hearing function in adult mice, and various gene-deficient mice show changes in morphogenesis of auditory ossicles in utero. Although murine auditory ossicles are tiny, their manipulation is feasible if one understands their anatomical orientation and 3D structure. Here, we describe how to dissect the auditory bulla and capsule of postnatal mice and then isolate individual ossicles by removing part of the bulla. We also discuss how to embed the bulla and capsule in different orientations to generate paraffin or frozen sections suitable for preparation of longitudinal, horizontal, or frontal sections of the malleus. Finally, we enumerate anatomical differences between mouse and human auditory ossicles. These methods would be useful in analyzing pathological, developmental and evolutionary aspects of auditory ossicles and the middle ear in mice.

We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis.

Dissektion der Auditory Bulla in postnatale Mäuse: Die Isolierung der Gehörknöchelchen und histologische Analyse

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-Clear: Ein Gewebe Clearing-Methode für die dreidimensionale Darstellung von Fettgewebe

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...