Name: reconstitution of &#946;-catenin degradation in xenopus egg extract
Uploaded: 2014-06-17T20:30:00
Description: Watch this Scientific Journal Video about Reconstitution Of ÃŽÂ²-catenin Degradation In Xenopus Egg Extract at JoVE.com

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).

1. Preparazione di Xenopus estratto Egg NOTA: Ogni rana produce circa 1 ml di estratto uovo utilizzabile. Estratti di 10 rane sono tipicamente preparati in una sola volta, e il volume di tampone di seguito descritto è per l&#39;esecuzione di un 10 rana Xenopus estratto uovo prep. Il volume buffer può essere regolata di conseguenza per preparazioni più o meno grandi di estratto uovo. Preps generato in questa maniera coerente produrre concentrazioni proteiche ≥ 50 mg / ml....

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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...

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.

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	<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>
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		<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>
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		<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>
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		<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 ÃŽÂ²-catenin Degradation In Xenopus Egg Extract at JoVE.com

Reconstitution Of ÃƒÅ½Ã‚Â²-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 ...

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

Unit 1

Translation: RNA to Protein

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Preparation and Fractionation of Xenopus laevis Egg Extracts

Preparazione e frazionamento di Xenopus laevis Egg Estratti

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

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Biologia

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

In Assemblea in vitro Nucleari Utilizzando frazionato Estratti Egg Xenopus

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Patch clamp e Tecniche di perfusione per la valutazione dei canali ioni Espresso in Xenopus Ovociti

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Studio del danno al DNA con Checkpoint<em&gt; Xenopus</em&gt; Uovo Estratti

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Produzione di<em&gt; Xenopus tropicalis</em&gt; Uovo Estratti per identificare RNA associata ai microtubuli

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

Facial Transplants in Xenopus laevis Embryos

Facial Transplants in Xenopus laevis Embryos

Trapianti viso a<em&gt; Xenopus laevis</em&gt; Embrioni

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

Il<em&gt; Xenopus</em&gt; Ovociti Cut-open vaselina Gap Tecnica Voltage-clamp Con fluorometria

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In Vitro Ricostituzione di Complessi Light-raccolta di piante e alghe verdi

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Quantification of Orofacial Phenotypes in Xenopus

Quantification of Orofacial Phenotypes in Xenopus

Quantificazione di orofacciale fenotipi in<em&gt; Xenopus</em

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

Ricostituzione funzionale e attività Canale Misure di purificata di tipo selvatico e mutante CFTR proteine

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...