Vorremmo ringraziare il laboratorio Lee dell&#39;Università del Minnesota per fornire i campioni di tessuto del midollo spinale del mouse che sono stati utilizzati. Questo lavoro è stato sostenuto dal Dipartimento della Difesa Ovarian Cancer Research Program (OCRP) OC093424 a MB, con la Ricerca sul Cancro e Fondo comunitario Randy Shaver a MB e dal Minnesota Ovarian Cancer Alliance (MOCA) a MB. I finanziatori avevano alcun ruolo nel disegno dello studio, la raccolta e l&#39;analisi dei dati, la decisione di pubblicare o preparazione del manoscritto.

1. Lysis Buffer Preparazione <ol><li> Sciogliere il saccarosio in acqua deionizzata (DI) acqua per fare una soluzione stock 2 M. Filtrare una soluzione 2 M di saccarosio con un aspirapolvere azionato 0,22 micron fluoruro di polivinilidene (PVDF) filtro. </li><li> Sciogliere ditiotreitolo (DTT) in acqua deionizzata per fare una soluzione stock di 500 mm e conservare in condizioni anaerobiche. Sciogliere il cloruro di magnesio (MgCl 2) in acqua deionizzata per fare una soluzione stock di 100 mm. Sciogliere adenosina trifosfato (ATP) disodio idrato in acqua deionizzata per fare una soluzione stock di 50 mm. </li><li> Portare una soluzione Tris a pH 7,4 sciogliendo Trizma cloridrato in acqua deionizzata e regolazione del pH usando idrossido di sodio (NaOH). </li><li> Combinare porzioni delle soluzioni madre in acqua deionizzata per fare un tampone di lisi con una concentrazione di saccarosio di 250 mM, una concentrazione di 1 mM DTT, una concentrazione MgCl 2 di 5 mM, una concentrazione di ATP di 1 mM e una concentrazione di 50 mM Tris . Ad esempio, miscelare 535,3 ml di Tris, 500 ml di MgCl 2, 1,25 ml di saccarosio, 20 l di DTT, 200 ml di ATP e 7,4947 ml di acqua deionizzata per fare 10 ml di tampone di lisi. Questo può essere usato per circa 20 esperimenti. </li></ol> 2. Le cellule in coltura per l&#39;esperimento <ol><li> Rendere i media aliquotandolo 30 ml di DMEM + 10% di siero fetale bovino (FBS) in un tubo da 50 ml. </li><li> Trasferire le cellule HeLa M17 o congelati dallo stoccaggio di azoto liquido a una piccola bicchiere con acqua tiepida. </li><li> Trasferire le cellule per provetta conica da 50 ml. In primo luogo, aggiungere alcuni media per la fiala cella. Poi il trasferimento delle cellule risospese entro 5 secondi di scongelamento. </li><li> Centrifugare la sospensione cellulare a 450 xg per 5 minuti per ottenere un pellet di cellule. Aggiungere 20 ml di media in un pallone T75. </li><li> Rimuovere il supporto da parte delle cellule con aspirazione. Aggiungere 5 ml di mezzi freschi per il pellet e risospendere. </li><li> Trasferire la sospensione 5 ml di cellule ai 20 ml di media nel pallone T75 e incubare a 37 ° C. Change media ogni 3 giorni. </li></ol> 3. La raccolta delle cellule <ol><li> Rimuovere il supporto tramite aspirazione, facendo attenzione a non toccare le cellule. </li><li> Lavare le cellule con 5 ml di tampone fosfato salino (PBS). Rimuovere PBS con aspirazione. </li><li> Aggiungere 3 ml di tripsina-acido etilendiamminotetraacetico (EDTA) al pallone T75 e incubare a 37 ° C per 3 min. </li><li> Aggiungere 6 ml di mezzi freschi al pallone e risospendere le cellule. </li><li> Trasferire la sospensione in un tubo da 15 ml e centrifugare a 290 xg per 5 minuti. </li><li> Rimuovere il surnatante e aggiungere 5 ml di PBS. Battere delicatamente la parte inferiore del tubo per rompere il pellet. Centrifugare a 290 xg per 5 min. </li><li> Ripetere il punto 3.6 a tre volte. </li><li> Rimuovere PBS e risospendere le cellule in 1 ml di PBS fresco. Trasferire al tubo Eppendorf (1,5 ml). Spin a 290 g per 5 min. </li></ol> 4. cellulare Lysis <ol><li> Misurare il volume approssimativo del pellet usando una pipetta und pesare perline di vetro in una massa: rapporto in volume di 1: 1. Aggiungere il doppio del volume del pellet in tampone di lisi. Nota: Aggiungere il buffer prima di aggiungere le perle di vetro. </li><li> Lisare le cellule nel tubo Eppendorf alla massima agitazione per 30 minuti a 4 ° C vortexando in una camera fredda. </li><li> Brevemente Centrifugare a 200 xg per 30 secondi per risolvere le perline. Raccogliere il surnatante. </li><li> Aggiungere lo stesso volume di perline di vetro come prima nel surnatante. </li><li> Vortex nuovamente alla massima agitazione per 30 minuti a 4 ° C. </li><li> Brevemente Centrifugare a 200 xg per 30 secondi per risolvere le perle di vetro. Raccogliere il surnatante. </li><li> Centrifugare a 5.030 xg per 5 minuti per rimuovere i nuclei, membrane e cellule intatte. Raccogliere il supernatante, che è il lisato cellulare. </li></ol> 5. Tissue omogeneizzazione <ol><li> Fai una massa: volume di 1: 9 soluzione di tessuto del midollo spinale del mouse per tampone di lisi. Nota: Questo metodo è applicabile ad una varietà di differirecampioni di tessuto Ent. </li><li> Omogeneizzare il tessuto utilizzando un&#39;impostazione del livello 2 sulla omogeneizzatore per 30 sec per garantire che non vi siano blocchi rimanenti. </li><li> Spin down a 5.030 xg per 3 min per rimuovere i nuclei, membrane e le cellule intatte. </li><li> Raccogliere il supernatante, che è il lisato. </li></ol> 6. Campione Derivatizzazione <ol><li> Eseguire un acido bicinconinico (BCA) in un&#39;analisi piastra a 96 pozzetti per determinare la concentrazione di proteine ​​del lisato tissutale utilizzando un lettore di piastre a 96 pozzetti colorimetrico come specificato dal protocollo del kit Protein Assay Pierce BCA. </li><li> Portare una aliquota corrispondente a 20 mg di proteine ​​totali di 50 microlitri utilizzando (DI) acqua deionizzata. </li><li> Aggiungere 2 ml di 1,35 mM di HA-Ub-Vinyl sulfone (VS) alla soluzione e incubare per 1 ora a 37 ° C. Ciò provoca 20 ug di lisato 50 nM di sonda nella miscela di reazione finale. Questo è in forte eccesso molare per assicurare la codifica di dubs funzionali. </li><li> IOncubate il campione in tampone campione di Laemmli per 5 minuti a 95 ° C. Per un 52 microlitri reazione volumi uso 26 ml di tampone 2x campione, 13 ml di tampone campione 4x o 8,87 ml di tampone campione 6x. </li><li> Raffreddare in ghiaccio prima di caricare il gel. </li></ol> 7. Western Blot <ol><li> Caricare un 12-pozzetti 4-20% gel tris-glicina con 40 ml di campione preparato </li><li> Attivare il gel a 95 V finché il fronte ione raggiunge il fondo. </li><li> Effettuare un trasferimento notturno del gel su un polivinilidene difluoruro (PVDF) membrana. </li><li> Incubare la membrana nel ammidico macchia nera e scansire il membane per ottenere un&#39;immagine che mostra la quantità di proteina in ogni corsia. </li><li> Decolorare la membrana e incubare 5% di latte in PBS per 1 ora per bloccare. </li><li> Incubare la membrana nella anticorpo anti-HA primario in una concentrazione di 1: 10.000 in 5% di latte in PBS o notte a 4 ° C o per 4 ore a temperatura ambiente. </li><li> Effettuare 3 lavaggi fo 5 min ciascuna in PBS con 0,1% di Tween-20 detergente. </li><li> Incubare la membrana in una perossidasi di rafano topo ad una concentrazione di 1: 10.000 in 5% di latte in PBS per almeno 2 ore. </li><li> Effettuare 3 lavaggi per 5 minuti ciascuna in PBS con 0,1% Tween-20 detergente. </li><li> Eseguire 1 lavaggio per 5 min in PBS. </li><li> Incubare la membrana in un reagente di rilevamento chemiluminescente per 10 min e rilevare utilizzando un rivelatore a chemiluminescenza. Utilizzare l&#39;impostazione sul rivelatore di esposizione automatica. </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>

Gli autori non hanno nulla da rivelare.

Dal momento che l&#39;ubiquitinazione è un attività cellulare fondamentale, la comprensione dei meccanismi di regolamentazione potrebbe essere la chiave per dissotterrare i processi di malattia e patologia. L&#39;uso di HA tagged sito diretto sonde attive Ub-derivati ​​qui riportati fornisce un metodo semplice, ma altamente applicabile per studiare la degradazione delle proteine ​​Ub-mediata. Questo metodo è più rapido e meno costoso di studiare ciascuno dei dubs singolarmente. I...

Il sistema ubiquitina-proteasoma (UPS) serve come una delle principali vie di degradazione in cellule di mammifero. Substrati legati per la degradazione nel proteasoma sono covalentemente contrassegnati con polimeri di ubiquitina (Ub) 1. Prima che il substrato bersaglio entra proteasoma per la degradazione, l&#39;etichetta poli-ubiquitina deve essere rimosso. Una classe di enzimi noti come deubiquitinating enzimi (dubs) è responsabile per la rimozione e il riciclaggio delle molecole di ubiquitina 2. E &#39;stato previsto dal genoma umano che ci sono quasi un centinaio dubs che lavorano nella cella 3. Con un gran numero di dubs che controllano processi cellulari Ub-mediate tale, lo studio di questi enzimi rappresenta una sfida in quanto le tecniche di mRNA non danno informazioni sull&#39;attività e western blotting dà solo informazioni sui livelli di espressione. L&#39;uso di emoagglutinina influenzale (HA) tagged, Ub-derived sito diretto sonde attive permette una covalente modification dei dubs funzionali e quindi dà una visualizzazione diretta delle attività di questi enzimi in un Western Blot 4. Le sonde hanno un gruppo tiolo reattivo C-terminale che funge da substrato suicida per il sito attivo residuo di cisteina 5. Con queste sonde, è possibile studiare l&#39;attività e potenziale espressione di molti DUBS sotto le due stati patologici e fisiologici della cellula. Le variazioni di attività DUB sono stati implicati in una serie di condizioni patologiche come il Parkinson, l&#39;Alzheimer, anemia e vari tipi di cancro 6-10. Questa tecnica fornisce un potente strumento per lo studio della malattia. In questo lavoro, mostriamo l&#39;applicazione di questa tecnica in cellule HeLa e M17 che sono state lisate usando perline di vetro. Inoltre, si delineano come utilizzare questo metodo in campioni di tessuto del midollo spinale del mouse. Le informazioni ottenute da questa tecnica può essere usato come punto di partenza per l&#39;identificazionebersagli terapeutici così come i modelli che stabiliscono per lo studio delle diverse condizioni di malattia. La vera utilità di questa tecnica risiede nella sua capacità di fornire informazioni su più dubs in un singolo dosaggio.

<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

Il sistema ubiquitina-proteasoma è stato recentemente coinvolto in diverse patologie, tra cui le malattie neurodegenerative e il cancro. Alla luce di questo, tecniche per studiare il meccanismo di regolazione di questo sistema sono essenziali per chiarire i processi cellulari e molecolari delle malattie summenzionate. L&#39;uso di emoagglutinina derivante sonde ubiquitina delineate nel presente documento è uno strumento prezioso per lo studio di questo sistema. Dettagli Questo documento un metodo che consente all&#39;utente di eseguire saggi che danno una visualizzazione diretta della deubiquitinating attività enzimatica. Enzimi Deubiquitinating controllano la degradazione del proteasoma e condividono un&#39;omologia funzionale ai loro siti attivi, che permette all&#39;utente di indagare l&#39;attività di diversi enzimi in un test. Lisati sono ottenuti attraverso dolce rottura delle cellule meccanico e incubate con sito attivo sonde dirette. Enzimi funzionali sono contrassegnati con le sonde mentre enzimi inattivi rimangono non legato. Di corsaning questo test, l&#39;utente ottiene informazioni sia l&#39;attività e potenziale espressione di molteplici enzimi deubiquitinating in modo facile e veloce. Il metodo attuale è molto più efficiente rispetto all&#39;utilizzo anticorpi individuali per i predetti cento enzimi deubiquitinating nella cellula umana.

Cellule HeLa M17 e coltivate sono state raccolte con il metodo descritto nel (cella di raccolta 3.) il protocollo e il tessuto del midollo spinale del mouse è stato ottenuto. Il / tessuto del midollo spinale pellet cellulare è stato posto in un tubo con il tampone di lisi descritto nella sezione di preparazione del reagente. Pellet cellulari sono stati lisati con perle di vetro (Figura 1A) e campioni di tessuto del midollo spinale di topo sono stati omogeneizzati usando l&#39;omogeneizzatore (...

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

Dettagli Il protocollo attuale un metodo per misurare l&#39;attività degli enzimi funzionalmente omologo deubiquitinating. Sonde specializzate covalentemente modificano l&#39;enzima e consentono il rilevamento. Questo metodo ha il potenziale di identificare nuovi bersagli terapeutici.

Metodo per la misurazione dell&#39;attività di Deubiquitinating enzimi in linee di cellule e tessuti Campioni

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

Research

JoVE Journal

Biology

9.5K Views.  University of Minnesota.  Dettagli Il protocollo attuale un metodo per misurare l'attività degli enzimi funzionalmente omologo deubiquitinating. Sonde specializzate covalentemente modificano l'enzima e consentono il rilevamento. Questo metodo ha il potenziale di identificare nuovi bersagli terapeutici. 

Video: 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.

Actina Co-sedimentazione di analisi, per l&#39;Analisi del Binding Protein a F-Actina

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

Ricerca

Biologia

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.

ΓH2AX quantificazione dei Foci, a campioni di tessuto

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.

Un test per misurare l&#39;attività di Escherichia coli Inducibile Lisina 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 dispositivo per il recupero di campioni crioconservati cadere accidentalmente nelle cisterne criogeniche

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.

misurazione<em&gt; In Vitro</em&gt; Attività ATPasi per enzimatica Caratterizzazione

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.

Confronto di tre diversi metodi per la determinazione proliferazione cellulare in Breast Cancer linee cellulari

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.

Misurazione dell'attività enzimatica della lattasi in laboratorio

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: Un metodo sezionamento migliorato per Recalcitrante tessuti vegetali Campioni

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.

La dissezione del Auditory Bulla nei topi postnatale: Isolamento delle ossa dell&#39;orecchio medio e l&#39;analisi istologica

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: Un tessuto metodo di compensazione per l'Imaging tridimensionale del tessuto adiposo

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