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 hjælp til denne undersøgelse. Dette arbejde blev støttet af NSF KARRIERE indrømme MCB-1054962 til ALA

1.. Forberedelse Store Apple Juice-agarplader <ol><li> For at gøre 4 plader, tilsættes 375 ml H 2 O, 11,25 g flyve agar og en omrørerstav til en 1.000 ml kolbe. Dette er Mix A. Autoklave Mix A med kolben låg løst loft på én 30 min-steriliseringscyklus til flydende varer. </li><li> Tilføj 125 ml æblejuice, 12,5 g tabellen sukker og en omrørerstav i et 500 ml bægerglas. Dette er Mix B. Heat Mix B på en opvarmet platform under omrøring, og temperaturen holdes på ca 70 ° C, indtil autoklavering a...

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E., Ptudor Mahowald, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+gene+required+for+assembly+of+the+germ+plasm+in+Drosophila+melanogaster.">a gene required for assembly of the germ plasm in Drosophila melanogaster.</a> Cell. 43, 97-104 (1985).</li><li>Thomson, T., Lasko, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+tudor+is+essential+for+polar+granule+assembly+and+pole+cell+specification,+but+not+for+posterior+patterning.">Drosophila tudor is essential for polar granule assembly and pole cell specification, but not for posterior patterning.</a> Genesis. 40, 164-170 (2004).</li><li>Arkov, A. 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Genes &amp; development. 23, 2742-2752 (2009).</li><li>Matthews, K. A., Goldstein, L. S. B., Fyrberg, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster:+Practical+Uses+in+Cell+and+Molecular+Biology.">Drosophila melanogaster: Practical Uses in Cell and Molecular Biology.</a> Methods in Cell Biology. 44, 13-32 (1995).</li><li>Sambrook, J., Russell, D. 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Formaldehyd har været almindeligt anvendt som et tværbindingsreagens for at identificere protein-protein-og protein-nukleinsyre-interaktioner. Sin gode opløselighed og cellemembranpermeabilitet sammen med kompatibilitet med downstream massespektrometri procedurer, gør formaldehyd en ideel kandidat agent for intracellulære tværbindingsbetingelser applikationer 3,15-17. Især blev det med succes brugt til at identificere mRNAer forbundet med Vasa, et kritisk kønsceller RNA helicase i Drosophila ...

Isolering af multisubunit protein forsamlinger og DNA-eller RNA-protein-komplekser er udført for at identificere protein komplekser, genomisk loci anerkendt af DNA-bindende regulatoriske proteiner eller RNA mål for RNA-bindende proteiner. Forskellige metoder tillader genom-dækkende identifikation af DNA-sites anerkendt af transkriptionsfaktorer eller kromatin proteiner (chip-seq) 1 og RNA-mål, der er forbundet med en given RNA-bindende protein (CLIP-seq) 2.. Bibliotekerne af RNA-afledte cDNA&#39;er eller DNA-mål så dybt sekventeret. Disse fremgangsmåder anvender kemiske eller UV-induceret krydsbinding at stabilisere komplekser efterfulgt af immunpræcipitering (IP) med et antistof mod et protein komponent af det undersøgte kompleks. Under udviklingen af ​​en organisme, mange protein komplekser dannes forbigående. Derfor er det afgørende at analysere sammensætningen og funktionen af disse komplekser in vivo for at forstå de molekylære mekanismer, der styrer development. Sådan en in vivo analyse ville være bedre in vitro fremgangsmåde, da det er næsten umuligt at gengive indfødte koncentrationer af interagerende komponenter og cellulære biokemiske miljø in vitro. Her demonstrerer vi en in vivo tilgang, som vi kunne bruge til at isolere store protein-komplekser fra Drosophila embryoner. I denne metode, er protein-komplekser i levende embryoner tværbundet med en lav koncentration af formaldehyd og efterfølgende protein-komplekser af interesse isoleres ved IP med et antistof mod et kendt element af komplekserne efterfulgt af gel-oprensning af komplekser og massespektrometrianalyse til identificere ukendte komplekse komponenter. Da formaldehyd er i stand til at gennemtrænge cellemembranen og har et tværbindende området 2,3-2,7 Å 3 kan proteinkomplekser tværbindes in vivo, og de ​​komplekse komponenter er tilbøjelige til at være tæt på hinanden. I denne artikel vil vibeskriver denne fremgangsmåde ved hjælp af isolering af Tudor (Tud) protein-kompleks som et eksempel. Tud er en germline protein, som er afgørende for kønscelleoverførsel udvikling 4-7. Dette protein indeholder 11 TuD domæner vides at interagere med denatureret argininer eller lysiner andre polypeptider 8-10. Tidligere har vi skabt en transgen Drosophila linje, der udtrykker HA-mærket funktionel Tud 5, og derfor er specifikke anti-HA antistof, der anvendes til at trække ned Tud kompleks efter krydsbinding. Foruden protein-protein-tværbindinger, kan formaldehyd generere nukleinsyre-protein-tværbindinger og anvendes i chip-seq eksperimenter. Endvidere i Drosophila, in vivo krydsbinding med formaldehyd har muliggjort identifikation af en RNA-mål af Vasa RNA helicase protein 11. Mens der i denne artikel vil vi beskrive en metode til in vivo tværbinding og purverifikation af protein-komplekser fra Drosophila embryoner, kan denne metode tilpasses til andre organismer og væv.

<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 processer styres af multisubunit protein-komplekser. Ofte disse komplekser dannes forbigående og kræver oprindelige miljø at samle. Derfor, for at identificere disse funktionelle protein-komplekser, er det vigtigt at stabilisere dem in vivo før cellelyse og efterfølgende oprensning. Her beskriver vi en metode, der anvendes til at isolere store bona fide protein-komplekser fra Drosophila embryoner. Denne metode er baseret på embryo permeabilisering og stabilisering af komplekserne inde i embryoner ved in vivo-tværbinding under anvendelse af en lav koncentration af formaldehyd, der let kan krydse cellemembranen. Efterfølgende proteinkomplekset interesse immunooprenset efterfulgt af geloprensning og analyseret ved massespektrometri. Vi illustrerer denne fremgangsmåde ved hjælp af oprensning af en Tudor-protein-kompleks, som er afgørende for kimcellelinje udvikling. Tudor er et stort protein, som indeholder flere Tudor domæner - smallmoduler, der interagerer med denatureret argininer eller lysiner af target-proteiner. Denne fremgangsmåde kan tilpasses til isolering af native protein-komplekser fra forskellige organismer og væv.

Effektiviteten af tværbinding og vellykket oprensning af det tværbundne Tud protein-kompleks blev analyseret ved SDS-PAGE på en 3% - 7% trin gel (illustreret i figur 1), efterfulgt af western blot (figur 2). Formålet med anvendelse af 3% - 7% trin gel baseret på den effektive adskillelse af tværbundet Tud protein kompleks fra de resterende ikke-krydsbundet Tud protein og koncentration af komplekset. Under vores in vivo tværbindingsbetingelser...

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

Multikomponenttekstilprodukter proteinkomplekser spiller afgørende roller i løbet af cellulære funktion og udvikling. Her beskriver vi en fremgangsmåde til at isolere native protein-komplekser fra Drosophila embryoner efter in vivo-tværbinding efterfulgt af oprensning af de tværbundne komplekser til efterfølgende struktur-funktions-analyse.

En In vivo Crosslinking tilgang til Isoler proteinkomplekser Fra Drosophila Embryoner

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

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Biology

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

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Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

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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 oprensning af kinesin fra Drosophila Embryoner

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