Name: an in vivo crosslinking approach to isolate protein complexes from drosophila embryos
Uploaded: 2014-04-23T21:00:00
Description: Watch this Scientific Journal Video about An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos at JoVE.com

Vi takker Jordan Davis, Yanyan Lin, Eric Schadler og Jimiao Zheng for deres teknisk hjelp med denne studien. Dette arbeidet ble støttet av NSF KARRIERE gi MCB-1054962 til ALA

En. Forbereder stort eple Juice-agar plater <ol><li> For å gjøre fire tallerkener, legg 375 ml H 2 O, 11.25 g fly agar og en rørepinne til en 1000 ml kolbe. Dette er Mix A. Autoclave Mix A med kolben lokket løst avkortet på en 30 min-steriliseringssyklus for flytende varer. </li><li> Legg 125 ml eplejuice, 12,5 g sukker og en rørepinne til et 500 ml begerglass. Dette er Blanding B. Varme Mix B på en oppvarmet plattform under omrøring og opprettholde temperaturen på ca 70 ° C inntil det autoklaveri...

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Formaldehyd har blitt vanlig benyttet som kryssbindings reagens for å identifisere protein-protein og protein-nukleinsyre interaksjoner. Dens god oppløselighet og cellemembranen permeabilitet, sammen med kompatibiliteten med nedstrøms massespektrometri prosedyrer, gjør formaldehyd en ideell kandidat agent for intracellulære crosslinking applikasjoner 3,15-17. Spesielt ble det med hell brukes til å identifisere mRNA forbundet med Vasa, en kritisk bakterie celle RNA heli i Drosophila 11.</su...

Isolering av multisubunit proteinsammensetningene og DNA-eller RNA-proteinkomplekser er utført for å identifisere proteinkomplekser, genomiske loci gjenkjent av DNA-bindende regulatoriske proteiner eller RNA-mål på RNA-bindende proteiner. Ulike metoder tillate genome-wide identifikasjon av DNA sider anerkjent av transkripsjonsfaktorer eller kromatin proteiner (chip-SEQ) for en og RNA mål knyttet til en gitt RNA-bindende protein (CLIP-seq) 2. Bibliotekene av RNA-avledet cDNAs eller DNA målene blir så dypt sekvensert. Disse metodene brukes kjemiske eller UV-indusert tverrbinding for å stabilisere komplekser fulgt av immunoutfelling (IP) med et antistoff mot et protein-komponenten av de studerte kompleks. Under utvikling av en organisme, mange protein-komplekser dannes forbigående. Derfor er det viktig å analysere sammensetningen og funksjonen til disse kompleksene in vivo for å forstå de molekylære mekanismer som styrer development. En slik in vivo-analyse ville være overlegne i forhold til in vitro metode siden det er praktisk talt umulig å reprodusere opprinnelige konsentrasjoner av de samvirkende komponenter og cellulær biokjemiske miljø in vitro. Her viser vi en in vivo tilnærming som vi lykkes med å bruke til å isolere store proteinkomplekser fra Drosophila embryoer. I denne metoden, blir proteinkomplekser i de levende embryoer tverrbundet med en lav konsentrasjon av formaldehyd og etterfølgende protein-komplekser av interesse blir isolert ved IP med et antistoff mot en kjent komponent av kompleksene, etterfulgt av gel-rensing av kompleksene og massespektrometri-analyse til identifisere ukjente komplekse komponenter. Siden formaldehyd er i stand til å trenge gjennom cellemembranen og har et tverrbindingsområde av 2,3 til 2,7 Å. 3, kan proteinkomplekser kan fornettes in vivo, og de ​​komplekse komponenter er sannsynlig å være nær hverandre. I denne artikkelen har vibeskrive denne metode med bruk av isolering av Tudor (Tud) protein-komplekset som et eksempel. Tud er en kimlinje protein som er essensielt for utvikling av kimlinje 4-7. Dette proteinet inneholder 11 Tud domener som interagerer med denaturert arginines eller lysines av andre polypeptider 8-10. Tidligere har vi generert en transgen Drosophila linje som uttrykker HA-merket funksjonell Tud 5 og derfor er spesifikk anti-HA antistoff brukes til å trekke ned Tud kompleks etter kryssbinding. I tillegg til protein-protein-tverrbindinger, kan formaldehyd generere nukleinsyre-protein-tverrbindinger, og som brukes i chip-seq eksperimenter. Videre, i Drosophila, in vivo kryssbinding med formaldehyd har muliggjort identifisering av en RNA target for Vasa RNA helicase protein 11. Mens i denne artikkelen beskriver vi en metode for in vivo kryssbinding og purification av protein komplekser fra Drosophila embryo, kan denne metoden være tilrettelagt for andre organismer og vev.

<table border="0" cellpadding="0" cellspacing="0" width="858">
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Drosophila agar</td>
			<td style="width:235px;">Lab Scientific (http://www.labscientific.com/)</td>
			<td style="width:119px;">FLY-8020-1</td>
			<td style="width:289px;">Do not autoclave the water and agar mix for prolonged period of time as it will cause the apple juice plates to become fragile</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Methyl 4-hydroxybenzoate</td>
			<td style="width:235px;">Sigma (http://www.sigmaaldrich.com/united-states.html)</td>
			<td style="width:119px;">H5501</td>
			<td style="width:289px;">Other name: TEGOSEPT</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Population cage</td>
			<td style="width:235px;">Flystuff (http://www.flystuff.com/)</td>
			<td style="width:119px;">59-104</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Fine nylon mesh</td>
			<td style="width:235px;">Flystuff (http://www.flystuff.com/)</td>
			<td style="width:119px;">57-102</td>
			<td style="width:289px;">When making the collection basket, cut the mesh slightly larger than the opening of the falcon tube cap to ensure a tight seal</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dounce homogenizer</td>
			<td style="width:235px;">Sigma (http://www.sigmaaldrich.com/united-states.html)</td>
			<td style="width:119px;">D8938-1SET</td>
			<td style="width:289px;">Chill the homogenizer on ice and prerinse with cold lysis buffer before use to prevent protein degradation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Protease inhibitor cocktail&#160;</td>
			<td style="width:235px;">Roche (http://www.rocheusa.com/portal/usa)</td>
			<td style="width:119px;">4693132001</td>
			<td style="width:289px;">PBS could be used to prepare concentrated protease inhibitor stock solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-HA agarose beads</td>
			<td style="width:235px;">MBL international (http://www.mblintl.com/)</td>
			<td style="width:119px;">561-8</td>
			<td style="width:289px;">The kit also includes HA-peptide and spin columns</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HA-peptide</td>
			<td style="width:235px;">MBL international (http://www.mblintl.com/)</td>
			<td style="width:119px;">561-8</td>
			<td style="width:289px;">Prepare to 2 mg/ml with PBS&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spin Column</td>
			<td style="width:235px;">MBL international (http://www.mblintl.com/)</td>
			<td style="width:119px;">561-8</td>
			<td style="width:289px;">Spin columns are included as part of the kit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Isopropanol</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">BP26324</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Triton X-100</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">BP151-500</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Heptane</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">H350-4</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">PBS</td>
			<td style="width:235px;">Invitrogen (https://www.lifetechnologies.com/us/en/home.html)</td>
			<td style="width:119px;">AM9625</td>
			<td style="width:289px;">Dilute from 10 X to 1 X with nanopure water before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Formaldehyde</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">BP531-500</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycine</td>
			<td style="width:235px;">BioRad (www.bio-rad.com/&#8206;)</td>
			<td style="width:119px;">161-0724</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SDS</td>
			<td style="width:235px;">BioRad (www.bio-rad.com/&#8206;)</td>
			<td style="width:119px;">161-0301</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Urea</td>
			<td style="width:235px;">BioRad (www.bio-rad.com/&#8206;)</td>
			<td style="width:119px;">161-0730</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Phenylmethanesulfonyl fluoride</td>
			<td style="width:235px;">Sigma (http://www.sigmaaldrich.com/united-states.html)</td>
			<td style="width:119px;">P7626-1G</td>
			<td style="width:289px;">Prepare 200 mM stock solution in isopropanol then dilute to working concentration of 2 mM in lysis buffer</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">IGEPAL CA-630</td>
			<td style="width:235px;">Sigma (http://www.sigmaaldrich.com/united-states.html)</td>
			<td style="width:119px;">I8896-50ML</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tween 20</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">BP337-100</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">15-ml tubes</td>
			<td style="width:235px;">USA Scientific (http://www.usascientific.com/)</td>
			<td style="width:119px;">1475-0511</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bleach</td>
			<td style="width:235px;">Clorox brand</td>
			<td style="width:119px;"></td>
			<td style="width:289px;">Dilute with equal volume of nanopure water to make 50% bleach</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">50-ml tubes</td>
			<td style="width:235px;">BD Biosciences (http://www.bdbiosciences.com/home.jsp)</td>
			<td style="width:119px;">352098</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Top layer sieve</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">04-884-1AK</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Bottom layer sieve</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">04-884-1BA</td>
			<td style="width:289px;"></td>
		</tr>
	</tbody>
</table>

an in vivo crosslinking approach to isolate protein complexes from drosophila embryos

Mange cellulære prosesser som er styrt av multisubunit proteinkomplekser. Ofte disse kompleksene danne forbigående og krever opprinnelige miljø å montere. Derfor, for å identifisere disse funksjonelle proteinkomplekser, er det viktig for å stabilisere dem in vivo før cellelysering, og etterfølgende rensing. Her beskriver vi en metode som brukes for å isolere store bona fide protein komplekser fra Drosophila embryoer. Denne metoden er basert på embryo permeabilization og stabilisering av de komplekser inne i embryoer ved in vivo tverrbinding ved hjelp av en lav konsentrasjon av formaldehyd, noe som lett kan krysse cellemembranen. Deretter blir proteinkompleks av interesse immunopurified etterfulgt av gel-rensing og analysert ved massespektrometri. Vi illustrerer denne fremgangsmåte ved hjelp av rensing av et Tudor proteinkompleks, som er avgjørende for kimlinje utvikling. Tudor er et stort protein, som inneholder flere Tudor domener - litenmoduler som samhandler med denaturert arginines eller lysines av target proteiner. Denne fremgangsmåte kan tilpasses for isolering av fremmedproteinkomplekser fra forskjellige organismer og vev.

Effektiviteten av tverrbinding og en vellykket rensing av den tverrbundne Tud proteinkompleks ble analysert ved SDS-PAGE på en 3% - 7% gel trinn (se figur 1), etterfulgt av western blot (Figur 2). Hensikten med å bruke 3% - 7% trinn gel er basert på den effektive separering av tverrbundet Tud proteinkomplekset fra den gjenværende ikke-kryssbundet Tud protein og konsentrasjonen av komplekset. Under våre in vivo tverrbindingsforhold som er nevnt ...

Watch this Scientific Journal Video about An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos at JoVE.com

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Multikomponent proteinkomplekser spiller avgjørende roller i cellulær funksjon og utvikling. Her beskriver vi en fremgangsmåte til å isolere opprinnelige proteinkomplekser fra Drosophila embryo etter in vivo tverrbinding etterfulgt av rensing av de tverrbundne komplekser for etterfølgende struktur-funksjonsanalyse.

En In vivo Crosslinking Approach å isolere protein komplekser Fra Drosophila Embryo

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

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An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

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The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

In-vivo Centrifugation of Drosophila Embryos

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Imaging Protein-protein Interactions in vivo

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Upright Imaging of Drosophila Embryos

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Imaging Cell Shape Change in Living Drosophila Embryos

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Isolation and Purification of Kinesin from Drosophila Embryos

Isolation and Purification of Kinesin from Drosophila Embryos

Isolering og rensing av kinesin fra Drosophila Embryoer

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...