Biz bu çalışma ile teknik yardım için Ürdün Davis, Yanyan Lin, Eric SCHADLER ve Jimiao Zheng teşekkür ederim. Bu çalışma ALA MCB-1054962 hibe NSF KARİYER tarafından desteklenen

1.. Büyük Elma Suyu-agar Plakaları hazırlanması <ol><li> 4 plakaları yapmak için, 1000 ml&#39;lik bir şişeye 375 ml 2 H, O, 11.25 g uçucu agar ve bir karıştırma çubuğu ilave edin. Bu gevşek sıvı mallar için tek bir 30 dakika sterilizasyon döngüsünde şapkalı şişe kapağı Mix A. otoklav Mix A. </li><li> 500 ml&#39;lik bir behere 125 ml elma suyu, 12.5 g sofra şekeri ve bir karıştırma çubuğu ilave edin. Karışım A ve otoklavlama işlemi bitene kadar yaklaşık 70 ° C&#39;de karıştırıldıktan ve sıcaklığı muhafaza ederken bu ısıtılmış bir platformda Mix B. ısı Mix B. </li><li> Mix tamamlanmasından sonra bir otoklav, A. yavaşça kombine karışımı karıştırma devam Mix Mix B transferi ve bunun 15 dakika boyunca soğumaya bırakın. Aşırı soğutma ve bir koruyucu eklenmeden önce neden agar katılaşma kaçının. </li><li> , Etanol içinde% 10 (ağırlık / hacim) metil 4-hidroksibenzoat, bir çözüm olun. Bu, koruyucu olarak hareket edecektir. Yukarıda elma suyu-agar karışımına solüsyonun 3.75 ml ilave edilir. Anothe karıştırıldıktan Ol5 dakika r. </li><li> Her 300 cm 2 plastik veya köpük tabak içine koruyucu ile 125 ml elma suyu agar karışımı dökün. Bu sırada oda sıcaklığma soğumaya ve katılaşmaya olsun. Dökme sırasında, hava kabarcıkları oluşturan kaçının. Not: elma suyu agar plakaları hemen kullanıma hazırdır. Net sarar ile kullanılmayan tabak örtün ve bir hafta kadar 4 ° C&#39;de saklayın. </li></ol> 2.. Drosophila Embriyo Toplama ve Ön çapraz bağlama tedavisi <ol><li> Embriyolar elde edilecek olan yaklaşık 40.000 ila 50.000 sinekler ile nüfus kafes yerleştirin. </li><li> Her iki levha 12 orta standart mısır unu-melas 125 ml içeren yapın ve her plaka üzerinde kurutuldu, fırıncı mayası içinde yaklaşık 4 gr serpin. Sinekleri şişmanlatıyor 48 saat boyunca 25 ° C inkübatör nüfus kafesin içine bu tabak yerleştirin. </li><li> 25 ° C sıcaklığa kadar ıstınız 2 elma suyu-agar plakaları Her biri üzerinde kurutuldu, fırıncı mayası içinde yaklaşık 1 gr serpinplakası. 0-1 hr-eski embriyoların toplamaya başlamak için nüfus kafes içinde 2 tabak yerleştirin. </li><li> 1 saat koleksiyonu sonunda plakalarını çıkartın. Koleksiyonun bir tur gerekirse 2 yeni plakalar ile değiştirin. </li><li> Plakaları kapak ve ince bir fırça ile hafifçe embriyolar tekrar süspansiyon yeterince su ekleyin. Paslanmaz çelik tel örgü yapılmış iki tabakalı bir elekten tekrar süspansiyon haline embriyolar dökün. Not: Üst tabaka embriyo ile icar ederken büyük bir böcek parçalarını toplayan orta gözenek boyutu (850 mikron) örgü yapılır. Alt tabaka maya ve su geçişine için verirken, embriyolar toplar ince örgü (75 mikron) yapılmıştır. </li><li> 50 ml&#39;lik bir tüp ve ince naylon örgü ile yapılan bir toplama sepeti içine toplanan embriyolar transfer ince boya fırça kullanın. Su ile kısaca embriyoların durulayın sonra kuruması için kağıt havlu üzerine örgü kurulayın. </li><li> 3 dakika boyunca hafif bir döndürme ile% 50 çamaşır suyu embriyolar bırakın. Tam durulayınly su ile kalan çamaşır suyu çıkarmak için. Kurulamak için kağıt havlu üzerinde örgü kurulayın. </li><li> (Bu, daha uzun bir süre 30 saniye sürmemelidir) 15 saniye boyunca hafifçe çalkalanarak izopropanol veya çok embriyo kümeleri arıza kadar dechorionated embriyolar bırakın. Iyice kurulamak için kağıt havlu üzerinde örgü kurulayın. </li><li> Yeniden asılmış onları tutmak için embriyolar üzerinde heptan akışı için bir pipet kullanırken 5 dakika boyunca heptanda embriyolar daldırın. Kurulamak için kağıt havlu üzerinde örgü kurulayın. </li><li> 3 dakika boyunca% 0.1 Triton X-100 (PBST) içeren fosfat tamponlu salin, pH 7.4 (PBS) akışı ile embriyolar durulayın. </li><li> Toplama sepeti sökün ve 10 ml PBST ihtiva eden bir 15 ml tüp örgü ile birlikte embriyolar aktarılır. Örgü kapalı embriyoların yıkamak için tüp hafifçe çevirin. </li><li> Tüpten kafes çıkarın. Dikey konumda tüp koyun ve embriyolar yerçekimiyle tüpün dibine çöker sağlar. Not: Yaklaşık 100-300embriyoların ul bir kafesten bir 1 saat toplama beklenmektedir. </li><li> PBST çıkarın ve hemen çapraz bağlama gerçekleştirmeye devam. </li></ol> Toplanan Embriyolar 3.. In vivo Çapraz <ol><li> Kullanımdan hemen önce, PBST içinde% 0.2 &#39;lik formaldehit hazırlayın. Embriyoların için formaldehit sabitleyici 10 ml eklenir ve 10 dakika için bir döner sallayıcı üzerinde hafif bir çalkalama ile 25 ° C&#39;de inkübe edin. Günün sonunda kullanılmayan fiksatif atın. </li><li> Çapraz bağlama reaksiyonu söndürün <ol><li> Çapraz bağlama sonunda, embriyolar tüpün dibine çöker sağlar. </li><li> Formaldehit çözeltisi çıkarın. Hemen çapraz bağlama, reaksiyonun sönmesi için PBST içinde 0.25 M glisin 10 ml ekleyin. 5 dakika için bir döner sallayıcı üzerinde hafif bir çalkalama ile 25 ° C&#39;de inkübe edin. </li><li> Embriyoların tüpün dibine çöker olsun. Söndürme çözeltisi çıkarın. PBST&#39;de 10 ml üç kez ile embriyolar yıkayın. </li><li> Tamamen P çıkarınBir pipet kullanarak TSİ yıkama solüsyonu. -80 ° C&#39;de kullanıma kadar saklayın embriyolar. </li></ol></li></ol> 4.. Protein Numune Hazırlama ve immüno-çökeltme <ol><li> 2 ml lisiz tampon maddesi içinde bir Dounce homojenleştirici kullanılarak 500 ul çapraz bağlanmış homojen embriyolar (0.5 M üre,% 0.01 SDS,% 2 Triton X-100, PBS içinde 2 mM fenilmetansülfonil florit [PMSF] ve proteaz inhibitörü kokteyli). Not: Aksi belirtilmediği sürece oda sıcaklığında bu ve aşağıdaki adımları uygulayın. </li><li> 1.5 ml santrifüj tüplerine lizat aktarın ve yavaşça ayrıntılı lizizi sağlamak için 15 dakika için bir döner platform üzerinde sallayın. Hücre yıkıntıları pelet haline getirildi, 5 dakika boyunca 16,000 x g&#39;de lizat santrifüj. Temiz bir 15 ml tüp içinde süpernatant kaydedin. </li><li> Anti-HA agaroz boncuklar süpernatana çamur 40 ul ilave edin ve 2.5 saat süreyle dönüşlü bir platform üzerinde inkübe edilir. </li><li> 5 dakika boyunca 2500 x g&#39;de santrifüjleme ile boncuk Pelet. Süpernatantı ama beğenme bırakıntüp içinde ximately 250 ul. Geri kalan süpernatan içinde yeniden süspanse edin ve boncuklar 10 mikron gözenek filtresi ile bir 1.5 ml döndürme kolonuna transfer. </li><li> 10 saniye boyunca 12,000 x g&#39;de santrifüj spin kolon ve akış atmak. </li><li> 200 ul yıkama tamponu (0.1 M glisin, 1 M NaCl, 1% IGEPAL CA-630,% 0.1 Tween-20, PBS) eklenerek ve boncukları yıkayın. 10 saniye boyunca 12,000 x g&#39;de santrifüj ve sütun yıkama atın. Yıkandıkları iki kez daha tekrarlayın. </li><li> Boncuklara 40 ul yıkama tamponu (2 mg / ml PBS içinde HA-peptid) eklenir ve yavaşça çalkalanarak her 5 dakika ile 15 dakika boyunca 37 ° C&#39;de inkübe edin. 10 saniye boyunca 12,000 x g&#39;de santrifüj ve sütun elüat toplamak. </li><li> Anti-HA antikoru kullanılarak western 13-blot analizi ile kompleks bir elüsyon kontrol edin. </li></ol>

<ol>
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Yazarlar ifşa hiçbir şey yok.

Formaldehit genellikle protein-protein, protein-nükleik asit etkileşimleri belirlemek için bir çapraz bağlama maddesi olarak kullanılmaktadır. Birlikte aşağı kütle spektrometresi prosedürleri ile uyumluluğu ile iyi çözünürlük ve hücre zarı geçirgenliği, hücre içi çapraz uygulamalar 3,15-17 için formaldehit ideal bir aday ajan olun. Özel olarak, başarılı bir şekilde Vasa, Drosophila 11 önemli bir germ hücre RNA helikaz ile ilişkili mRNA tespit etmek için k...

Multisubunit protein montajlar ve DNA-ya da RNA-protein komplekslerinin izolasyonu protein kompleksleri, DNA-bağlanma düzenleyici proteinler veya RNA bağlama proteinlerinin RNA hedefleri tarafından kabul genomik lokusları tespit için gerçekleştirilir. Farklı yöntemler, belirli bir RNA-bağlama proteini (CLIP-seq) 2 ile ilişkili transkripsiyon faktörleri veya kromatin proteinleri (ChIP-seq) 1 ve RNA hedefleri tarafından tanınan DNA siteleri genom tanımlanmasını sağlar. RNA türetilmiş cDNA ya da DNA hedeflerinin diziler daha sonra derin dizilir. Bu yöntemler, kimyasal veya UV kaynaklı çalışılan kompleksinin bir protein bileşenine karşı bir antikor ile immüno-çökeltme (IP) takip kompleksleri stabilize etmek için çapraz bağlama kullanın. Bir organizmanın gelişimi sırasında, çok sayıda geçici protein kompleksleri oluşturur. Bu nedenle, kontrol dev moleküler mekanizmaların anlaşılması, in vivo olarak, bu komplekslerin bileşimi ve işlevini analiz için çok önemlidirelopment. Bu etkileşim bileşenleri ve in vitro olarak hücre biyokimyasal ortamı yerel konsantrasyonları üretmek için neredeyse imkansız olduğu için bu tür bir in vivo analiz in vitro yaklaşımına göre daha üstün olacaktır. Burada biz başarılı Drosophila embriyolar büyük protein kompleksleri izole etmek için kullanabileceğiniz bir in vivo yaklaşım göstermektedir. Bu yöntemde, canlı embriyolar protein kompleksleri düşük formaldehit konsantrasyonu ve daha sonra ilgi protein kompleksleri için kompleksleri ve kütle spektrometre analizi jel geçirilmiş, ardından arıtma komplekslerinin bilinen bir bileşenine karşı bir antikor ile IP ile izole edilir ile çapraz bağlanmış olan bilinmeyen karmaşık bileşenleri tanımlar. Formaldehit hücre zarı nüfuz edebilir ve 2.3-2.7 Â 3 bir dizi çapraz bağlama sahip olduğu için, protein kompleksleri, in vivo olarak çapraz bağlanabilir ve karmaşık parçaları birbirine yakın olması muhtemeldir. Bu makalede bizBir örnek olarak Tudor (Tud) protein kompleksinin izole edilmesini kullanarak bu yöntem açıklanmaktadır. Tud tohum çizgisi 4-7 gelişimi için gerekli olan bir tohum çizgisi proteindir. Bu protein için diğer polipeptidlerin 8-10 metillenmiş arginines veya lisin ile etkileşim için bilinen 11 Tud alanları içerir. Daha önce, fonksiyonel Tud 5 HA-etiketli ifade eder ve bu nedenle, spesifik bir anti-HA antikoru çapraz bağlamadan sonra Tud kompleksi aşağı çekmek için kullanılan bir transgenik Drosophila satır elde ettik. Protein-protein çapraz bağlar ek olarak, nükleik asit, formaldehit-protein çapraz bağlar oluşturabilir ve yonga-seq deneylerde kullanılır. Ayrıca, Drosophila, formaldehid ile çapraz bağlama, in vivo Vasa RNA helikaz protein 11 bir RNA hedefinin belirlenmesini sağladı. Bu yazıda in vivo çapraz bağlama ve pur için bir yöntem tarif ederkenDrosophila embriyolardan protein kompleksleri ification, bu yöntem diğer organizmalar ve dokular için uyarlanabilir.

<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

Pek çok hücresel süreçleri multisubunit protein kompleksleri tarafından kontrol edilir. Sıkça bu kompleksler geçici oluşturmak ve birleştirmek için doğal bir ortam gerektirir. Bu nedenle, bu işlevsel protein kompleksleri tespit etmek için, hücre lizizi ve daha sonra saflaştırma daha önce in vivo olarak bunları stabilize etmek için önemlidir. Burada Drosophila embriyolardan büyük niyetli protein komplekslerini izole etmek için kullanılan bir yöntem tarif eder. Bu yöntem, hücre zarını kolayca geçebilir formaldehit düşük bir konsantrasyonu kullanılarak in vivo çapraz bağlama ile embriyoların içine komplekslerin embriyo nüfuziyet ve stabilizasyon dayanmaktadır. Daha sonra, ilgilenilen protein kompleksi jel geçirilmiş, ardından arıtma ve kütle spektrometresi ile analiz edilir İmmüno. Bu tohum çizgisi gelişimi için gerekli olan bir Tudor protein kompleksi, saflaştırılmasını kullanarak bu yöntemi örneklemektedir. - Küçük Tudor birden Tudor etki alanı içeren büyük bir proteindirHedef proteinlerin metillenmiş arginines veya lisin ile etkileşim modülleri. Bu yöntem, farklı organizmalardan ve dokulardan yerli protein komplekslerinin izolasyonu için uyarlanabilir.

Western blot ve ardından (Şekil 1 &#39;de gösterilen)% 7 aşama jel (Şekil 2) -, çapraz bağlanma etkinliği ve çapraz bağlanmış Tud protein kompleksinin başarılı bir saflaştırma% 3 üzerinde SDS-PAGE ile analiz edildi. % 3 kullanmanın amacı, -% 7 adım jel kompleksinin geri kalan çapraz bağlanmamış Tud protein ve konsantrasyondan Tud çapraz bağlanmış protein kompleksinin etkili ayrılmasına dayanmaktadır. Yukarıda sözü edilen <e...

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

Çok bileşenli protein kompleksleri hücre fonksiyonu ve gelişme sırasında çok önemli bir rol oynamaktadır. Burada çapraz bağlayıcı in vivo daha sonra yapı-işlev analizi için çapraz bağlı kompleksler, ardından arıtma ardından Drosophila embriyolardan yerli protein komplekslerini izole etmek için kullanılan bir yöntem tarif eder.

Bir In vivo</emŞu Protein Kompleksler yalıtmak için> Çapraz yaklaşım Drosophila Embriyolar

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

an-vivo-crosslinking-approach-to-isolate-protein-complexes-from

Research

JoVE Journal

Biology

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

16.6K Görüntüleme.  Murray State University.  Çok bileşenli protein kompleksleri hücre fonksiyonu ve gelişme sırasında çok önemli bir rol oynamaktadır. Burada çapraz bağlayıcı in vivo daha sonra yapı-işlev analizi için çapraz bağlı kompleksler, ardından arıtma ardından Drosophila embriyolardan yerli protein komplekslerini izole etmek için kullanılan bir yöntem tarif eder. 

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

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Nöronal Kültürler Midgastrula Sahne Drosophila Embriyolar hazırlanması

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Biyoloji

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated through myosin-actin interactions and play an important role in our physiology.  During development, they enable cells to move from one location to the next in order to form the early structures of tissue.  Traction forces help in the healing processes.  They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body.  These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor.  The most common method by which to study cells in vitro has been to use a glass or polystyrene dish.  However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces.  Our lab has developed a technique to overcome these limitations.  The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process.  The pillars we use are 3 &mu;m in diameter, 10 &mu;m tall, and are configured in a regular array with 9 &mu;m center-to-center spacing.  But these physical dimensions can be readily varied to accommodate a variety of studies.  We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS.  We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections.  We call these substrates microfabricated post-array-detectors, or mPADs.  Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.

Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces

In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility.

Microfabricated Post-Dizi-Dedektörleri (mPADs): Mekanik Kuvvetleri yalıtın bir Yaklaşım

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated ...

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

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

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

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Elektron Mikroskobu için embriyo Hazırlık Drosophila Embriyonik kalp tüpü

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

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

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

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

Dayanıksız Multi-protein Kompleksleri izolasyonu In vivo Kontrollü Hücresel Çapraz Bağlama ve immuno-manyetik Afinite Kromatografisi

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

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

In-vivo Drosophila Embriyolar Santrifüj

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

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

Görüntüleme Protein-protein etkileşimleri In vivo</em

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

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

VE Dik Görüntüleme Drosophila Embriyolar

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

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Birincil Hücre Kültürleri Drosophila Gastrula Embriyolar

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

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

Living Görüntüleme Hücre Şekli Değiştir Drosophila Embriyolar

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

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Kinesin İzolasyonu ve Saflaştırılması gelen Drosophila Embriyolar

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

Bir In vivo Çapraz yaklaşım Drosophila Embriyolar