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

Vi takker Laurie Lee for kritisk lesing av manuskriptet. TWC er støttet av en American Heart Association Predoctoral Fellowship (12PRE6590007). MRB er støttet av en National Cancer Institute trening stipend (T32 CA119925). SSH er støttet av National Institutes of Health (R01DK078640). EL er støttet av National Institutes of Health (R01GM081635 og R01GM103926).

En. Utarbeidelse av Xenopus Egg Extract MERK: Hver frosk gir ca 1 ml av brukbare egg ekstrakt. Utdrag fra 10 frosker er vanligvis forberedt på en gang, og volumet av buffer beskrevet nedenfor er for å utføre en 10 frosk Xenopus egg ekstrakt prep. Buffervolumet kan bli justert tilsvarende for større eller mindre produkter av egg ekstrakt. Forbereder generert på denne måten konsekvent gi proteinkonsentrasjoner ≥ 50 mg / ml. Fremgangsmåte for innsamling av egg og behand...

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Xenopus egg ekstrakt er en robust biokjemisk system for å undersøke β-catenin omsetning. Konsentrasjonen av β-catenin i Xenopus egg ekstrakt er ca 25 nM 2. Under optimale betingelser er det egg-ekstrakt kan nedbryte β-catenin med en hastighet på 50 til 100 nM / hr, og er halv-maksimal ved 200 nM 24. Det er flere viktige skritt for vellykket tilberedning av β-catenin degradering bruker Xenopus egg ekstrakt. Disse omfatter 1) å generere høykvalitets Xenopus</em...

Xenopus laevis egg ekstrakt har vært brukt mye til å studere mange cellebiologiske prosesser, inkludert cytoskeletal dynamikk, kjernefysisk sammenstilling og import, apoptose, ubiquitin metabolisme, cellesyklus progresjon, signaltransduksjon, og protein omsetning 1-17. Den Xenopus egg ekstrakt system er mottagelig for den biokjemiske analysen av en hærskare av cellulære prosesser fordi egg ekstrakt representerer i hovedsak ufortynnet cytoplasma som inneholder alle de essensielle cytoplasmatiske komponentene som er nødvendige for å utføre disse prosessene og gjøre det mulig etterforskning. Store mengder egg ekstrakt kan fremstilles på en gang for biokjemiske manipulasjoner som krever store mengder av materiale (f.eks, proteinrensing eller high-throughput screening) 18-20. En annen fordel er at konsentrasjonen av spesifikke proteiner i Xenopus egg ekstrakt som kan nøyaktig innstilles ved tilsetning av rekombinant protein og / eller immunodepletion av endoggent proteiner i motsetning til transfeksjon av plasmid-DNA, hvor ekspresjon av proteinet av interesse er vanskelig å kontrollere. I tillegg, kan mangelen på tilgjengelige rekombinante proteiner kan overvinnes ved tilsetning av transkripter som koder for proteinet av interesse ved å utnytte den nylagede Xenopus egg ekstrakt med høy kapasitet for å oversette eksogent tilsatt mRNA. Reguleringen av protein degradering er kritisk for kontroll av mange cellulære stier og behandler 21. Xenopus egg ekstrakt har vært brukt mye for å studere protein degradering som systemet gir mulighet for flere måter å overvåke protein omsetning uten konfunderende påvirkninger av transkripsjon og oversettelse. Wnt signalveien er en høyt konservert signalveien som spiller avgjørende roller i utvikling og sykdom. Omsetningen av β-catenin, den viktigste effektor av Wnt veien, er sterkt regulert, og en økt steady-state lEvel av β-catenin er kritisk for aktivering av Wnt målgener. Betydningen av β-catenin degradering er markert med det faktum at mutasjoner i Wnt veien som hemmer β-catenin nedbrytning funnet i ~ 90% av alle sporadiske tilfeller av tykk-og endetarmskreft 22. β-catenin nedbrytning av komponenter i Wnt veien kan trofast rekapitulert i Xenopus egg ekstrakt for å studere mekanismen for omsetningen, så vel som for å identifisere nye små molekyl modulatorer av dens nedbrytning 2, 19, 20, 23-29. Fremgangsmåter for fremstilling av Xenopus egg ekstrakt for å studere cellesyklus er blitt beskrevet i tidligere publikasjoner JOVE 30-32. Den aktuelle protokoll beskriver en modifikasjon av disse fremgangsmåter, og er optimalisert for degradering av [35 S]-radioaktivt merket β-catenin, og luciferase-merket β-catenin iXenopus egg ekstrakt. Den radiomerket degradering analysen gir mulighet for direkte visualisering av proteinnivåer via autoradiografi. [35 S] metionin er innlemmet i proteinet av interesse ved å bruke en in vitro oversettelses reaksjon som kan så bli direkte tilsatt til en nedbrytningsreaksjon. I tillegg vil det radio-merkede protein turn assay krever ikke et antistoff mot proteinet av interesse, eller en epitop tag, som kan påvirke proteinstabilitet. Fordi selv små endringer i proteinnivåer, noe som reflekteres i forandringer i intensiteten av det radioaktivt merkede proteinbånd, fremstilles lett visualisert ved autoradiografi, at [35 S]-radioaktivt merket nedbrytning assay representerer en meget nyttig metode for visualisering av protein turn 2. Fusjon av β-catenin til ildflue luciferase (heretter referert til som bare &quot;luciferase&quot;) gjør det mulig for nøyaktige og lettvinte kvantitative målinger av proteinnivåer, forfor å bestemme de kinetiske egenskaper av β-catenin omsetning 19, 20.. En stor fordel av luciferase-analysen er at den gir en sterk kvantitativ system som er lett skaleres opp. Den følgende protokollen gir enkle metoder for å analysere β-catenin nedbrytning, og en robust, effektiv og effektiv metode for high-throughput screening av nye β-catenin modulatorer.

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			<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>
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		<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>
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		</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 egg ekstrakt er et godt karakterisert, robust system for å studere biokjemi av diverse cellulære prosesser. Xenopus egg ekstrakt er blitt brukt til å studere protein omsetning i mange cellulære sammenhenger, inkludert cellesyklus-og signaloverføringsbaner 1-3. Heri er en fremgangsmåte beskrevet for å isolere Xenopus egg ekstrakt som har blitt optimalisert for å fremme nedbrytning av den kritiske Wnt veien komponent, β-catenin. To forskjellige metoder er beskrevet for å vurdere β-catenin proteindegradering i Xenopus egg ekstrakt. En metode er visuelt rikt ([35 S]-radioaktivt merkede proteiner), mens den andre er lettere skalert for high-throughput-analyser (ildflueluciferase-merkede fusjonsproteiner). De teknikker som er beskrevet kan brukes til, men er ikke begrenset til, vurdere β-catenin protein turn og identifisere molekylære komponenter som bidrar til omsetningen. I tillegg er ABILligheten for å rense store mengder homogen Xenopus egg ekstrakt kombinert med kvantitative og lettvinte avlesning av luciferase-merket proteiner gjør at dette systemet skal være lett tilpasses for high-throughput screening for modulatorer av β-catenin degradering.

En skjematisk β-catenin nedbrytning i Xenopus egg ekstrakt er vist i figur 2A. 35. S-merkede β-catenin ble inkubert i Xenopus egg ekstrakt, ble aliquoter (1 ml ekstrakt ekvivalent) fjernes til riktig tid, og prøver ble underkastet SDS-PAGE etterfulgt av autoradiografi. β-catenin nedbrytning av komponenter i Wnt veien er mediert av ubiquitin-proteasom-systemet 2, og degradering av [35 S]-radioaktivt merket β-catenin i Xenopus ekstrakt inhib...

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

En fremgangsmåte er beskrevet for å analysere proteinnedbrytning ved hjelp av radioaktivt merket og luciferase-fusjonsproteiner i Xenopus egg ekstrakt og dens tilpasning for high-throughput screening for lite molekyl modulatorer av proteinnedbrytning.

Rekonstituering av β-catenin degradering i<em&gt; Xenopus</em&gt; Egg Extract

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

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

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

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

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

Studie av DNA Damage Checkpoint hjelp<em&gt; Xenopus</em&gt; Egg ekstrakter

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

Produksjon av<em&gt; Xenopus tropicalis</em&gt; Egg Ekstrakter å identifisere microtubule-tilknyttede RNA

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

Ansikts transplantasjoner i<em&gt; Xenopus laevis</em&gt; Embryo

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

Den<em&gt; Xenopus</em&gt; Oocytter Cut-open vaselin Gap Voltage-klemme teknikk med fluorometri

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 Rekonstituering of Light innhøsting Komplekser av planter og grønne alger

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

Kvantifisering av Orofacial fenotyper i<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

Funksjonell tilberedning og Channel Aktivitet Målinger av Renset villtype og Mutant CFTR Protein

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