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 Î²-catenin Degradation In Xenopus Egg Extract at JoVE.com

Vi tackar Laurie Lee för kritisk läsning av manuskriptet. TWC stöds av en American Heart Association Predoctoral Fellowship (12PRE6590007). MRB stöds av en National Cancer Institute utbildningsbidrag (T32 CA119925). SSH stöds av National Institutes of Health (R01DK078640). EL stöds av National Institutes of Health (R01GM081635 och R01GM103926).

1. Framställning av Xenopus äggextrakt OBS: Varje groda ger ungefär 1 ml användbar ägg extrakt. Extrakt från 10 grodor framställs typiskt vid en tid, och den volym buffert som beskrivs nedan är för att utföra en 10 grodan Xenopus äggextrakt prep. Buffertvolymen kan justeras i enlighet därmed för större eller mindre beredningar av ägg extrakt. Preps alstras på detta sätt genomgående bildning av proteinkoncentrationer ≥ 50 mg / ml. Processen att samla in äg...

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Xenopus ägg Extrakt är en robust biokemiskt system för att utreda β-catenin omsättning. Koncentrationen av β-catenin i Xenopus äggextrakt är ~ 25 nM 2. Under optimala betingelser är äggextrakt förmåga att bryta ned β-catenin med en hastighet av 50 till 100 nm / h och är halv-maximal vid 200 nM 24. Det finns flera viktiga steg för lyckad beredning av β-catenin nedbrytning med hjälp av Xenopus ägg extrakt. Dessa inkluderar en) alstra högkvalitativt Xe...

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

Xenopus laevis ägg extrakt har använts i stor utsträckning för att studera många cellbiologiska processer inklusive cytoskelettala dynamik, kärn montering och import, apoptos, ubiquitin metabolism, cellcykelreglering, signaltransduktion och proteinomsättningen 1-17. Xenopus äggextrakt systemet är mottaglig för den biokemiska analysen av en legion av cellulära processer eftersom ägg extrakt representerar i huvudsak outspädd cytoplasma som innehåller alla viktiga cytoplasma komponenter som krävs för att genomföra dessa processer och möjliggör utredning. Stora mängder ägg extrakt kan förberedas på en gång för biokemiska manipulationer som kräver stora mängder material (t.ex. proteinrening eller high-throughput screening) 18-20. En annan fördel är att koncentrationen av specifika proteiner i Xenopus äggextrakt kan justeras exakt genom tillsats av rekombinant protein och / eller immunotömning av endogenous proteiner i motsats till transfektion av plasmid-DNA, där expression av proteinet av intresse är svårt att kontrollera. Dessutom kan avsaknaden av tillgängliga rekombinanta proteiner övervinnas genom tillsats av transkript som kodar för proteinet av intresse och dra fördel av den nyberedda Xenopus äggextrakt höga kapacitet att översätta exogent tillsatta mRNA. Regleringen av proteinnedbrytning är avgörande för kontrollen av många cellulära vägar och processer 21. Xenopus ägg extrakt har använts i stor utsträckning för att studera proteinnedbrytning som systemet tillåter flera sätt att övervaka proteinomsättningen utan confounding influenser av transkription och translation. Den Wnt signalväg är ett starkt konservativa signalväg som spelar avgörande roller i utveckling och sjukdom. Omsättningen av β-catenin, den stora effektor av den Wnt banan, är hårt reglerad, och en ökad jämvikts lEvel av β-catenin är kritiskt för aktivering av Wnt mål gener. Vikten av β-catenin nedbrytning markeras av att mutationer i den Wnt banan som hämmar β-catenin nedbrytning som finns i ~ 90% av alla sporadiska fall av kolorektal cancer 22. β-catenin nedbrytning av komponenter i den Wnt banan kan troget rekapituleras i Xenopus ägg extrakt för att studera mekanismen av omsättningen samt att identifiera nya småmolekylära modulatorer av dess nedbrytning 2, 19, 20, 23-29. Metoder för framställning av Xenopus äggextrakt för att studera cellcykeln har beskrivits i tidigare jove publikationer 30-32. Den nuvarande protokollet beskriver en modifiering av dessa metoder och är optimerad för nedbrytningen av [35S]-märkt β-catenin och luciferas-taggade β-catenin iXenopus ägg extrakt. Analys Den radiomärkta nedbrytning möjliggör direkt visualisering av proteinnivåer genom autoradiografi. [35 S] metionin införlivas i proteinet av intresse med användning av en in vitro-translationsreaktion som sedan kan tillsättas direkt till ett reaktions nedbrytning. Dessutom tar analys den radioaktivt märkta proteinomsättning inte kräva en antikropp mot proteinet av intresse eller en epitopmärkning, som kan påverka proteinstabilitet. Därför att även små förändringar i proteinnivåer, vilket avspeglas i förändringar i intensiteten av det radioaktivt märkta proteinband, lätt visualiseras genom autoradiografi, den [35 S]-märkt analys nedbrytning utgör en mycket användbar metod för visualisering av proteinomsättningen 2. Fusion av β-catenin till eldfluga luciferas (nedan kallat bara &quot;luciferas&quot;) möjliggör exakta och facile kvantitativa mätningar av proteinnivåer, i syfteatt bestämma de kinetiska egenskaperna hos β-catenin omsättning 19, 20. En stor fördel med den luciferas-analysen är att det ger en stark kvantitativ system som lätt skalas upp. Följande protokoll ger enkla metoder för analys av β-catenin nedbrytning och en robust, effektiv och effektiv metod för high-throughput screening av nya β-catenin modulatorer.

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			<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>
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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>
		<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 ägg extrakt är en väldefinierad, robust system för att studera biokemi olika cellulära processer. Xenopus ägg extrakt har använts för att studera proteinomsättningen i många cellulära sammanhang, bland annat cellcykeln och signaltransduktionsvägar 1-3. Häri beskrivs ett förfarande för isolering av Xenopus äggextrakt som har optimerats för att främja nedbrytningen av den kritiska Wnt banan komponent, β-catenin. Två olika metoder har beskrivits för att utvärdera β-catenin proteinnedbrytning i Xenopus äggextrakt. En metod är visuellt informativa ([35S]-märkt protein), medan den andra är mer lätt skalas för high-throughput analyser (eldflugeluciferas-märkta fusionsproteiner). De tekniker som beskrivs kan användas för, men är inte begränsade till, bedöma β-catenin proteinomsättning och identifiera molekylära komponenter som bidrar till dess omsättning. Dessutom fartyhet för att rena stora mängder homogen Xenopus äggextrakt kombination med den kvantitativa och enkel avläsning av luciferas-taggade proteiner tillåter detta system för att enkelt anpassas för high-throughput screening för modulatorer av β-catenin nedbrytning.

En schematisk bild av β-catenin nedbrytning i Xenopus äggextrakt visas i figur 2A. 35 S-märkt β-catenin inkuberades i Xenopus äggextrakt alikvoter (1 ml extrakt ekvivalent) avlägsnas vid lämpliga tidpunkter, och prover utsattes för SDS-PAGE följt av autoradiografi. β-catenin nedbrytning av komponenterna i den Wnt banan förmedlas av ubiquitin-proteasome systemet 2, och nedbrytning av [35S]-märkt β-catenin i Xenopus extrakt inhiberas...

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

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

Ett förfarande beskrivs för att analysera proteinnedbrytning med användning av radiomärkt och luciferas-fusionsproteiner i Xenopus äggextrakt och dess anpassning för high-throughput screening för småmolekylära modulatorer av proteinnedbrytning.

Rekonstitution av β-catenin nedbrytning i<em&gt; Xenopus</em&gt; Egg Utdrag

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

SCIENTISTS IN ACTION

Preparation and Fractionation of Xenopus laevis Egg Extracts

Upprättande och fraktionering av Xenopus laevis Egg Utdrag

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

In Vitro Nuclear Assembly Använda Fraktionerade Xenopus Egg Utdrag

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 och tekniker perfusion för att studera jonkanaler Uttryckt i Xenopus Oocyter

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 med<em&gt; Xenopus</em&gt; Egg Extrakt

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

Produktion av<em&gt; Xenopus tropicalis</em&gt; Ägg extrakt för att identifiera mikrotubuli-associerade 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 Transplantationer i<em&gt; Xenopus laevis</em&gt; Embryon

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; Äggcellen Cut-öppen vaselin Gap spänning-clamp teknik med fluorometri

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

Quantification of Orofacial Phenotypes in Xenopus

Quantification of Orofacial Phenotypes in Xenopus

Kvantifiering av Orofacial fenotyper 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

Funktionell beredning och Kanalaktivitets Mätningar av renat Vildtyps och Mutant CFTR Protein

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

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Cykloheximid Chase Analys av proteinnedbrytning i<em&gt; Saccharomyces cerevisiae</em

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein ...