Ringraziamo Laurie Lee per la lettura critica del manoscritto. TWC è supportato da un American Heart Association Predoctoral Fellowship (12PRE6590007). MRB è sostenuto da una borsa di formazione del National Cancer Institute (T32 CA119925). SSH è supportato dal National Institutes of Health (R01DK078640). EL è supportato dal National Institutes of Health (R01GM081635 e R01GM103926).

<ol>
	<li>Glotzer, M., Murray, A. W., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclin+is+degraded+by+the+ubiquitin+pathway.">Cyclin is degraded by the ubiquitin pathway.</a> Nature. 349, 132-138 (1991).</li><li>Salic, A., Lee, E., Mayer, L., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+beta-catenin+stability:+reconstitution+of+the+cytoplasmic+steps+of+the+wnt+pathway+in+Xenopus+egg+extracts.">Control of beta-catenin stability: reconstitution of the cytoplasmic steps of the wnt pathway in Xenopus egg extracts.</a> Molecular Cell. 5, 523-532 (2000).</li><li>Murray, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+extracts.">Cell cycle extracts.</a> Methods in cell biology. 36, 581-605 (1991).</li><li>Dabauvalle, M. C., Scheer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+nuclear+pore+complexes+in+Xenopus+egg+extract.">Assembly of nuclear pore complexes in Xenopus egg extract.</a> Biology of the cell / under the auspices of the European Cell Biology Organization. 72, 25-29 (1991).</li><li>Tutter, A. V., Walter, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+DNA+replication+in+a+soluble+cell-free+system+derived+from+Xenopus+eggs.">Chromosomal DNA replication in a soluble cell-free system derived from Xenopus eggs.</a> Methods Mol Biol. 322, 121-137 (2006).</li><li>Theriot, J. A., Rosenblatt, J., Portnoy, D. A., Goldschmidt-Clermont, P. J., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+profilin+in+the+actin-based+motility+of+L.+monocytogenes+in+cells+and+in+cell-free+extracts.">Involvement of profilin in the actin-based motility of L. monocytogenes in cells and in cell-free extracts.</a> Cell. 76, 505-517 (1994).</li><li>Maresca, T. J., Heald, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+studying+spindle+assembly+and+chromosome+condensation+in+Xenopus+egg+extracts.">Methods for studying spindle assembly and chromosome condensation in Xenopus egg extracts.</a> Methods Mol Biol. 322, 459-474 (2006).</li><li>Shennan, K. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+egg+extracts:+a+model+system+to+study+proprotein+convertases.">Xenopus egg extracts: a model system to study proprotein convertases.</a> Methods Mol Biol. 322, 199-212 (2006).</li><li>Kornbluth, S., Yang, J., Powers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+cell+cycle+using+Xenopus+egg+extracts.">Analysis of the cell cycle using Xenopus egg extracts.</a> Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]. 11, (2006).</li><li>Chan, R. C., Forbes, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+study+of+nuclear+assembly+and+nuclear+import+using+Xenopus+egg+extracts.">In vitro study of nuclear assembly and nuclear import using Xenopus egg extracts.</a> Methods Mol Biol. 322, 289-300 (2006).</li><li>Masui, Y., Markert, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+control+of+nuclear+behavior+during+meiotic+maturation+of+frog+oocytes.">Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes.</a> The Journal of experimental zoology. 177, 129-145 (1971).</li><li>Forbes, D. J., Kirschner, M. W., Newport, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+formation+of+nucleus-like+structures+around+bacteriophage+DNA+microinjected+into+Xenopus+eggs.">Spontaneous formation of nucleus-like structures around bacteriophage DNA microinjected into Xenopus eggs.</a> Cell. 34, 13-23 (1983).</li><li>Lohka, M. J., Masui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+in+vitro+of+sperm+pronuclei+and+mitotic+chromosomes+induced+by+amphibian+ooplasmic+components.">Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components.</a> Science. 220, 719-721 (1983).</li><li>Newport, J. W., Kirschner, M. W. <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+the+cell+cycle+during+early+Xenopus+development.">Regulation of the cell cycle during early Xenopus development.</a> Cell. 37, 731-742 (1984).</li><li>Mitchison, T., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+instability+of+microtubule+growth.">Dynamic instability of microtubule growth.</a> Nature. 312, 237-242 (1984).</li><li>Blow, J. J., Laskey, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+DNA+replication+in+nuclei+and+purified+DNA+by+a+cell-free+extract+of+Xenopus+eggs.">Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs.</a> Cell. 47, 577-587 (1986).</li><li>Verma, R., 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=Ubistatins+inhibit+proteasome-dependent+degradation+by+binding+the+ubiquitin+chain.">Ubistatins inhibit proteasome-dependent degradation by binding the ubiquitin chain.</a> Science. 306, 117-120 (2004).</li><li>Yu, H., King, R. W., Peters, J. M., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+ubiquitin-conjugating+enzyme+involved+in+mitotic+cyclin+degradation.">Identification of a novel ubiquitin-conjugating enzyme involved in mitotic cyclin degradation.</a> Curr Biol. 6, 455-466 (1996).</li><li>Thorne, C. 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=Small-molecule+inhibition+of+Wnt+signaling+through+activation+of+casein+kinase+1alpha.">Small-molecule inhibition of Wnt signaling through activation of casein kinase 1alpha.</a> Nature Chemical Biology. 6, 829-836 (2010).</li><li>Thorne, C. 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=A+biochemical+screen+for+identification+of+small-molecule+regulators+of+the+Wnt+pathway+using+Xenopus+egg+extracts.">A biochemical screen for identification of small-molecule regulators of the Wnt pathway using Xenopus egg extracts.</a> Journal of Biomolecular Screening. 16, 995-1006 (2011).</li><li>Hinkson, I. V., Elias, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+state+of+protein+turnover:+It's+about+time.">The dynamic state of protein turnover: It's about time.</a> Trends in Cell Biology. 21, 293-303 (2011).</li><li>Kinzler, K. W., Vogelstein, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lessons+from+Hereditary+Colorectal+Cancer.">Lessons from Hereditary Colorectal Cancer.</a> Cell. 87, 159-170 (1996).</li><li>Lee, E., Salic, A., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+regulation+of+[beta]-catenin+stability+by+Tcf3+and+CK1epsilon.">Physiological regulation of [beta]-catenin stability by Tcf3 and CK1epsilon.</a> J Cell Biol. 154, 983-993 (2001).</li><li>Lee, E., Salic, A., Kr&#252;ger, R., Heinrich, R., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+APC+and+Axin+derived+from+experimental+and+theoretical+analysis+of+the+Wnt+pathway.">The roles of APC and Axin derived from experimental and theoretical analysis of the Wnt pathway.</a> PLoS Biology. 1, (2003).</li><li>Seeling, J. 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=Regulation+of+beta-catenin+signaling+by+the+B56+subunit+of+protein+phosphatase+2A.">Regulation of beta-catenin signaling by the B56 subunit of protein phosphatase 2A.</a> Science. 283, 2089-2091 (1999).</li><li>Guger, K. A., Gumbiner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-Catenin+has+Wnt-like+activity+and+mimics+the+Nieuwkoop+signaling+center+in+Xenopus+dorsal-ventral+patterning.">beta-Catenin has Wnt-like activity and mimics the Nieuwkoop signaling center in Xenopus dorsal-ventral patterning.</a> Dev Biol. 172, 115-125 (1995).</li><li>Cselenyi, C. S., 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=LRP6+transduces+a+canonical+Wnt+signal+independently+of+Axin+degradation+by+inhibiting+GSK3's+phosphorylation+of+&#946;-catenin.">LRP6 transduces a canonical Wnt signal independently of Axin degradation by inhibiting GSK3's phosphorylation of &#946;-catenin.</a> Proceedings of the National Academy of Sciences. 105, 8032-8037 (2008).</li><li>Jernigan, K. 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=Gbetagamma+activates+GSK3+to+promote+LRP6-mediated+beta-catenin+transcriptional+activity.">Gbetagamma activates GSK3 to promote LRP6-mediated beta-catenin transcriptional activity.</a> Science Signaling. 3, (2010).</li><li>Major, M. B., 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=Wilms+tumor+suppressor+WTX+negatively+regulates+WNT/beta-catenin+signaling.">Wilms tumor suppressor WTX negatively regulates WNT/beta-catenin signaling.</a> Science. 316, 1043-1046 (2007).</li><li>Cross, M. K., Powers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obtaining+eggs+from+Xenopus+laevis+females.">Obtaining eggs from Xenopus laevis females.</a> J Vis Exp. , (2008).</li><li>Cross, M. K., Powers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+fractionation+of+Xenopus+laevis+egg+extracts.">Preparation and fractionation of Xenopus laevis egg extracts.</a> J Vis Exp. , (2008).</li><li>Willis, J., DeStephanis, D., Patel, Y., Gowda, V., Yan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+DNA+damage+checkpoint+using+Xenopus+egg+extracts.">Study of the DNA damage checkpoint using Xenopus egg extracts.</a> J Vis Exp. , (2012).</li><li>Sive, H. L., Grainger, R. M., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Early Development of Xenopus Laevis : A Laboratory Manual. , (2000).</li><li>Rubinfeld, B., 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=Binding+of+GSK3&#946;+to+the+APC-&#946;-Catenin+Complex+and+Regulation+of+Complex+Assembly.">Binding of GSK3&#946; to the APC-&#946;-Catenin Complex and Regulation of Complex Assembly.</a> Science. 272, 1023-1026 (1996).</li><li>Salic, A., King, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+small+molecule+inhibitors+of+the+ubiquitin-proteasome+pathway+in+Xenopus+egg+extracts.">Identifying small molecule inhibitors of the ubiquitin-proteasome pathway in Xenopus egg extracts.</a> Methods in Enzymology. 399, 567-585 (2005).</li><li>Trinkle-Mulcahy, 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=Identifying+specific+protein+interaction+partners+using+quantitative+mass+spectrometry+and+bead+proteomes.">Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes.</a> J Cell Biol. 183, 223-239 (2008).</li><li>Tan, C. 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=Wnt+signalling+pathway+parameters+for+mammalian+cells.">Wnt signalling pathway parameters for mammalian cells.</a> PLoS One. 7, (2012).</li></ol>

Gli autori dichiarano di non avere interessi finanziari in competizione.

Estratto uovo di Xenopus è un sistema biochimico robusto per indagare fatturato β-catenina. La concentrazione di β-catenina in estratto uovo di Xenopus è ~ 25 Nm 2. In condizioni ottimali, l&#39;estratto uovo è in grado di degradare β-catenina ad una velocità di 50-100 nM / hr e metà-massimale a 200 nM 24. Ci sono diversi passaggi critici per il successo ricostituzione del degrado β-catenina con estratto di uova di Xenopus. Questi includono 1) generare Xenopu...

A correction was made to <a href="http://www.jove.com/video/51425">Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract</a>. At the time of publication there were some instances where an incorrect volume notation was used. These instances were corrected from:
2.1.2. Add extract to 1/10 the volume of pelleted antibody or affinity beads (e.g., 20 ml pelleted beads to 200 ml extract). In order to minimize dilution of the extract, withdraw as much liquid from the beads as possible before addition of the extract using gel loading tips with long, tapered tips.
2.2.5. Aliquot the appropriate volumes for degradation assay into pre-chilled microfuge tubes on ice. For radiolabeled &#946;-catenin degradation assays, withdraw 2-5 ml extract for each time point.
3.2.3. At the designated time point, remove 1-5 ml of the sample and mix immediately with SDS sample buffer (5x volume) to stop the reaction. To make sure the degradation reaction is completely terminated, flick tube several times and vortex vigorously.
3.2.4. Perform SDS-PAGE/autoradiography. Run 1 ml equivalents (~50 mg of protein) of the extract for each time point/lane. Degradation of &#946;-catenin in Xenopus egg extract should be evidenced by the time-dependent decrease in intensity of the radiolabeled &#946;-catenin band Figure 2. Quantify results using ImageJ, ImageQuant, or other preferred imaging software if necessary.
4.2.2. Add in vitro-translated &#946;-catenin-luciferase fusion (from 4.1) into prepared Xenopus reaction mix (from 2.2) on ice and mix well as in 3.2.1. NOTE: The activity of the &#946;-catenin luciferase that is added to the extract is typically between 20 - 50,000 relative luminescence units (RLU)/ml of extract (based on measurements obtained from 4.1.2). Starting signal should be approximately 100,000 RLU (2-5 ml of the in vitro-translated &#946;-catenin-luciferase fusion).
to:
2.1.2. Add extract to 1/10 the volume of pelleted antibody or affinity beads (e.g., 20 &#181;l pelleted beads to 200 &#181;l extract). In order to minimize dilution of the extract, withdraw as much liquid from the beads as possible before addition of the extract using gel loading tips with long, tapered tips.
2.2.5. Aliquot the appropriate volumes for degradation assay into pre-chilled microfuge tubes on ice. For radiolabeled &#946;-catenin degradation assays, withdraw 2-5 &#181;l extract for each time point.
3.2.3. At the designated time point, remove 1-5 &#181;l of the sample and mix immediately with SDS sample buffer (5x volume) to stop the reaction. To make sure the degradation reaction is completely terminated, flick tube several times and vortex vigorously.
3.2.4. Perform SDS-PAGE/autoradiography. Run 1 &#181;l equivalents (~50 mg of protein) of the extract for each time point/lane. Degradation of &#946;-catenin in Xenopus egg extract should be evidenced by the time-dependent decrease in intensity of the radiolabeled &#946;-catenin band Figure 2. Quantify results using ImageJ, ImageQuant, or other preferred imaging software if necessary.
4.2.2. Add in vitro-translated &#946;-catenin-luciferase fusion (from 4.1) into prepared Xenopus reaction mix (from 2.2) on ice and mix well as in 3.2.1. NOTE: The activity of the &#946;-catenin luciferase that is added to the extract is typically between 20 - 50,000 relative luminescence units (RLU)/&#181;l of extract (based on measurements obtained from 4.1.2). Starting signal should be approximately 100,000 RLU (2-5 &#181;l of the in vitro-translated &#946;-catenin-luciferase fusion).

Erratum: Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Estratto uovo Xenopus laevis è stato ampiamente utilizzato per studiare molti cellulari processi biologici, tra cui la dinamica del citoscheletro, l&#39;assemblaggio e l&#39;importazione nucleare, l&#39;apoptosi, il metabolismo ubiquitina, progressione del ciclo cellulare, trasduzione del segnale, e proteine ​​fatturato 1-17. Il sistema estratto uovo Xenopus è riconducibile alla analisi biochimica di una legione di processi cellulari perché estratto uovo rappresenta sostanzialmente citoplasma non diluito che contiene tutti i componenti essenziali citoplasmatici necessari per eseguire questi processi e consentire indagine. Grandi quantità di estratto di uova possono essere preparati in una sola volta per le manipolazioni biochimiche che richiedono grandi quantità di materiale (ad esempio, purificazione di proteine ​​o high-throughput screening) 18-20. Un altro vantaggio è che la concentrazione di proteine ​​specifiche in estratto uovo Xenopus può essere regolata con precisione mediante aggiunta di proteine ​​e / o immunodeplezione di endog ricombinanteproteine ​​enous in contrasto trasfezione di DNA plasmidico in cui l&#39;espressione della proteina di interesse è difficile da controllare. Inoltre, la mancanza di proteine ​​ricombinanti disponibili può essere superato con l&#39;aggiunta di trascritti codificanti la proteina di interesse, sfruttando elevata capacità estratto uovo Xenopus preparata per tradurre mRNA esogenamente aggiunti. La regolazione della degradazione delle proteine ​​è fondamentale per il controllo di molte vie cellulari ed elabora 21. Estratto uovo di Xenopus è stato ampiamente utilizzato per studiare la degradazione delle proteine ​​in quanto il sistema permette diversi modi per monitorare il turnover proteico, senza influenze confondenti di trascrizione e traduzione. La via di segnalazione Wnt è una via di segnalazione altamente conservato che svolge un ruolo critico nello sviluppo e nella malattia. Il fatturato di β-catenina, il principale effettore della via di Wnt, è altamente regolamentato, e un aumento steady-state level di β-catenina è critica per l&#39;attivazione di Wnt geni bersaglio. L&#39;importanza di degradazione β-catenina è evidenziata dal fatto che mutazioni nella via di Wnt che inibiscono la degradazione β-catenina trovare in ~ 90% di tutti i casi sporadici di tumore colorettale 22. β-catenina degradazione di componenti della via di Wnt può essere fedelmente riassunta in estratto uovo Xenopus per studiare il meccanismo del fatturato nonché per identificare nuovi modulatori piccole molecola della sua degradazione 2, 19, 20, 23-29. Metodi per la preparazione di estratto uovo Xenopus per studiare il ciclo cellulare sono stati descritti in precedenti pubblicazioni JoVE 30-32. Il protocollo attuale descrive una modifica di questi metodi ed è ottimizzato per la degradazione di [35 S]-radiomarcato β-catenina e luciferasi-tag β-catenina inEstratto uovo di Xenopus. Il test di degradazione radioattivo permette la visualizzazione diretta dei livelli di proteine ​​tramite autoradiografia. [35 S] metionina è incorporata nella proteina di interesse utilizzando una reazione di traduzione in vitro che possono poi essere aggiunto direttamente ad una reazione di degradazione. Inoltre, la proteina radiomarcato dosaggio fatturato non richiede un anticorpo contro la proteina di interesse o un epitopo, che può influenzare la stabilità della proteina. Poiché anche piccoli cambiamenti nei livelli di proteine, che si riflette in variazioni dell&#39;intensità della banda di proteina radiomarcato, sono facilmente visualizzati mediante autoradiografia, la [35 S]-radiomarcato saggio degradazione rappresenta un metodo molto utile per la visualizzazione di proteine ​​fatturato 2. Fusione di β-catenina a luciferasi di lucciola (di seguito denominato semplicemente &quot;luciferasi&quot;) consente di misure quantitative precise e facili dei livelli di proteine, al fine diper determinare le proprietà cinetiche di β-catenina fatturato 19, 20. Uno dei principali vantaggi del saggio luciferasi è che fornisce un sistema quantitativo forte che è facilmente calcificato. Il protocollo che segue fornisce metodi semplici per saggiare la degradazione β-catenina e un metodo affidabile, efficiente ed efficace per high-throughput screening nuovi modulatori β-catenina.

<table border="0" cellpadding="0" cellspacing="0" style="width:709px;" width="710">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Material</td>
			<td style="width:132px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:243px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pregnant Mare Serum Gonadotropin (PMSG)</td>
			<td style="width:132px;">ProSpec</td>
			<td style="width:119px;">hor-272-A</td>
			<td>Reconstituted with distilled water before use.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Human Chorionic Gonadotropin</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">CG10-10VL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Potassium Chloride</td>
			<td style="width:132px;">Fisher</td>
			<td style="width:119px;">BP366-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium Chloride</td>
			<td style="width:132px;">Research Products International</td>
			<td style="width:119px;">S23020-5000.0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium Chloride</td>
			<td style="width:132px;">Fisher</td>
			<td style="width:119px;">BP214-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcium Chloride</td>
			<td style="width:132px;">Acros Organics</td>
			<td style="width:119px;">AC42352-5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES</td>
			<td style="width:132px;">Fisher</td>
			<td style="width:119px;">BP310-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cysteine</td>
			<td style="width:132px;">Acros Organics</td>
			<td style="width:119px;">AC17360-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Leupeptin</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">L2884-10MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aprotinin</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">A1153-10MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pepstatin</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">P4265-5MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cytochalasin B</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">C8273-10MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">3 mL syringe: Luer Lock tip</td>
			<td style="width:132px;">Becton Dickinson</td>
			<td style="width:119px;">309657</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">27G1 needle</td>
			<td style="width:132px;">Becton Dickinson</td>
			<td style="width:119px;">305109</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">96 well solid white polystyrene microplate, round bottomed</td>
			<td style="width:132px;">Corning</td>
			<td style="width:119px;">3605</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Steady-Glo Luciferase assay system</td>
			<td style="width:132px;">Promega</td>
			<td style="width:119px;">E2520</td>
			<td>Store long-term at -80, can store for up to 1 month at -20</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TNT Sp6 coupled reticulocyte lysate system</td>
			<td style="width:132px;">Promega</td>
			<td style="width:119px;">L4600</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">TNT Sp6 High-yield Wheat Germ protein expression system</td>
			<td style="width:132px;">Promega</td>
			<td style="width:119px;">L3260</td>
			<td>Generally higher yield than reticulocyte lysate&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">EasyTag Express Protein Labeling Mix [S35]</td>
			<td style="width:132px;">Perkin Elmer</td>
			<td style="width:119px;">NEG772007MC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Creatine phosphate</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">27920-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ATP</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">A2383-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Creatine phosphokinase</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">C3755-35KU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dimethyl Sulfoxide (DMSO)</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">D8418-50ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dual-Glo luciferase assay system</td>
			<td style="width:132px;">Promega</td>
			<td style="width:119px;">E2920</td>
			<td>Same storage conditions as Steady-Glo</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 mL Centrifuge tubes</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:119px;">0556214D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sorvall SS-34 fixed angle rotor</td>
			<td style="width:132px;">Thermo Scientific</td>
			<td style="width:119px;">28020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">115 V 50/60 Hz Minicentrifuge</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:119px;">05-090-128</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mMessage mMachine Sp6 kit</td>
			<td style="width:132px;">Ambion</td>
			<td style="width:119px;">AM1340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Firefly Luciferase antibody</td>
			<td style="width:132px;">Abcam</td>
			<td style="width:119px;">ab16466</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-GSK3 antibody</td>
			<td style="width:132px;">BD Transduction Laboratories</td>
			<td style="width:119px;">610201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:132px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:132px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:132px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:132px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Equipment</td>
			<td style="width:132px;">Company</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FLUOStar Optima</td>
			<td style="width:132px;">BMG Labtech</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sorvall RC-6 Plus Centrifuge</td>
			<td style="width:132px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">16&#176;C Incubator</td>
			<td style="width:132px;">Percival Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

reconstitution of &#946;-catenin degradation in xenopus egg extract

Xenopus laevis estratto uovo è un ben caratterizzato, robusto sistema per studiare la biochimica di diversi processi cellulari. Estratto uovo Xenopus è stato usato per studiare il ricambio proteico in molti contesti cellulari, tra cui il ciclo cellulare e trasduzione del segnale percorsi 1-3. Qui, è descritto un metodo per isolare estratto uovo Xenopus che è stato ottimizzato per promuovere la degradazione della componente pathway Wnt critico, β-catenina. Due diversi metodi sono descritti per valutare β-catenina degradazione delle proteine ​​in estratti di uova di Xenopus. Un metodo è visivamente informativo ([35 S] radiomarcato proteine), mentre l&#39;altro è più facilmente scalato per saggi high-throughput (lucciola proteine ​​di fusione luciferasi-tagged). Le tecniche descritte possono essere utilizzate per, ma non sono limitati a, valutare β-catenina turnover proteico e identificare i componenti molecolari che contribuiscono al fatturato. Inoltre, la poslità di purificare grandi volumi di omogenea estratto uovo Xenopus combinata con la lettura quantitativa e facile di proteine ​​luciferasi-tag permette questo sistema di essere facilmente adattato per screening ad alto rendimento per modulatori di degradazione β-catenina.

Uno schema di degradazione β-catenina in estratto uovo Xenopus è mostrato in Figura 2A. 35 S-marcato β-catenina è stata incubata in estratto uovo Xenopus, aliquote (1 ml estratto equivalente) sono stati rimossi al momento opportuno, ed i campioni sono stati sottoposti a SDS-PAGE seguita da autoradiografia. β-catenina degradazione di componenti della via di Wnt è mediata dal sistema ubiquitina-proteasoma 2, e il degrado di [35 S]-radiomarcato β-ca...

Watch this Scientific Journal Video about Reconstitution Of Î²-catenin Degradation In Xenopus Egg Extract at JoVE.com

Reconstitution Of &amp;#946;-catenin Degradation In Xenopus Egg Extract

Un metodo è descritto per l&#39;analisi degradazione delle proteine ​​usando radioattivo e proteine ​​luciferasi-fusione in estratto uovo Xenopus e il suo adattamento per lo screening high-throughput per piccoli modulatori molecola di degradazione delle proteine.

Ricostituzione della β-catenina degradazione nel<em&gt; Xenopus</em&gt; Estrai Egg

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

reconstitution-of-catenin-degradation-in-xenopus-egg-extract

Research

Education

JoVE Journal

JoVE Core

Molecular Biology

Biology

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Unit 1

Translation: RNA to Protein

SCIENTISTS IN ACTION

12.0K Views.  Vanderbilt University Medical Center.  Un metodo è descritto per l'analisi degradazione delle proteine ​​usando radioattivo e proteine ​​luciferasi-fusione in estratto uovo Xenopus e il suo adattamento per lo screening high-throughput per piccoli modulatori molecola di degradazione delle proteine. 