This work was supported by a grant from the Canadian Institutes for Health Research (MOP-86514).

Fareler ile Deneysel işlemler McGill Üniversitesi (protokol 4966) Hayvan Bakım Komitesi tarafından kabul edildi. Kültürlenmiş Hücrelerde elde edilen protein ekstrelerinin hazırlanması 1. <ol><li> Buz soğukluğunda fosfat tamponlu tuzlu su içinde 10 ml (PBS) ile iki kez kültürlenmiş hücreleri yıkayın. </li><li> Kauçuk bir ya da 1.5 ml mikrosantrifüj tüpü içine buzla soğutulmuş PBS transfer süspansiyon 1 ml&#39;lik plastik bir hücre kazıyıcı ile ya yapışmış olan hücrelerin Pençe. </li><li> 4 ° C&#39;de 700 x g&#39;de 5 dakika boyunca bir mikrosantrifüj içinde spin. Aspire PBS. </li><li> 10 7 hücre başına buz soğukluğunda sitoplazmik lizis tamponu içinde 100 ul (Tablo 1) ekleyin ve yukarı ve aşağı pipet ve. </li><li> 20 dakika boyunca buz üzerinde inkübe edilir. </li><li> 4 ° C&#39;da bir mikrosantrifüj içinde tam hızda 10 dk için spin. </li><li> Atın pelet. Yeni 1.5 ml mikrosantrifüj tüp içine süpernatant aktarın ve buz üzerinde tutmak. </li><li> DetBradford tahlili kullanılarak 23 - (10 ug / ml, genellikle 1) ermin protein konsantrasyonu. </li><li> Kullanılana kadar -80 ° C&#39;de kısım ve mağaza hücre ekstrelerinden oluşabilir. </li></ol> Fare karaciğer ve dalak elde edilen protein ekstrelerinin 2. Hazırlık <ol><li> CO 2 inhalasyon ile bir fare Euthanize. </li><li> Diseksiyon tahta üzerinde temiz bir pad üzerinde ötenazi hayvan yatırın. Makas ile karın açın. </li><li> Makas ve forseps kullanılarak karaciğer ve dalak ayır ve yaklaşık 50 ml buz gibi soğuk PBS içinde, her doku yıkayın. </li><li> Hemen bir neşter ile küçük parçalar halinde dokuları kesmek (örneğin: Yaklaşık 1-2 mm 3). </li><li> Gecikme olmadan, taze cryotube dokuların parçaları koymak ve daha sonra sıvı azot içinde bunları dondurmak oturtun. Mağaza şekild dondurulmuş doku alikotlan kullanılana kadar -80 ° C&#39;de ilave edildi. </li><li> 0.5 ml - 0.25 - (2 ila 3 mm, yaklaşık 1) dondurulmuş dokudan bir parça homojenizebuz soğukluğunda sitoplazmik lizis tamponu (Tablo 1) 10 saniye süreyle bir doku homojenleştirici ile. </li><li> 20 dakika boyunca buz üzerinde 1.5 ml mikrosantrifüj tüpü ve soğuk Homojenat aktarın. </li><li> 4 ° C&#39;da bir mikrosantrifüj içinde tam hızda 10 dk için spin. </li><li> Yeni 1.5 ml mikrosantrifüj tüp içine pelet ve transfer süpernatant atın. Buz üzerinde tutun. </li><li> Bradford tahlili 23 kullanılarak - (10 ug / ul genellikle 1) protein konsantrasyonu belirleyin. </li><li> Kullanılana kadar -80 ° C&#39;de kısım ve mağaza hücre ekstrelerinden oluşabilir. </li></ol> Radyoaktif IRE-prob 3. Hazırlık <ol><li> Kısıtlama endonükleaz Xbal (plazmid ug başına 1 U) DDE sekansının alt baş ayrıştıran ile 1 saat boyunca 37 ° C&#39;de inkübe edilerek İRE içeren plazmid I-12.CAT 22 linearize. linearize plazmid, in vitro transkripsiyonu için, şablon olarak kullanılır. </li><li> Kurman, in vitro olarak 20 ul bir toplam hacim içinde transkripsiyon reaksiyonu. Tablo 2&#39;de gösterilen stok çözümleri kullanın ve ekleyin: 1 ul çizgiselleştirilmiş plazmid şablonu, 4 ul transkripsiyon tamponu; ATP / TO / GTP karışımı 1 ul karışımı, 10 ul [32 P α-] -UTP, 2 ul ditiyotreitol, 1 ul RNaz inhibitör ve 1 ul, T7 RNA polimeraz ile karıştırıldı. Yukarı ve aşağı pipetleme karıştırın. </li><li> 1 saat 24 süreyle 40 ° C&#39;de inkübe edilir. </li></ol> Radyoaktif IRE-prob 4. saflaştırılması <ol><li> Yukarı ve aşağı pipetleme 0.5 M EDTA, pH 8 Karışımı 1 ul ekleyerek in vitro transkripsiyon reaksiyonu bitirin. </li><li> Daha iyi çökelmesi için taşıyıcı olarak, 10 mg / ml tRNA 10 ul ekle. Yukarı ve aşağı pipetleme karıştırın. </li><li> 3 M amonyum asetat, 82.5 ul ekleyin. Vorteks ile karıştırın. </li><li> Etanol içinde 273 ul ekle. Vorteks ile karıştırın. </li><li> 5 dakika boyunca oda sıcaklığında bekletin. </li><li> Oda sıcaklığında bir mikrosantrifüj içinde tam hızda 10 dakika boyunca dönerler. Sil süpernatant. </li><li> Yıkama,% 70 etanol içinde 100 ul ile pelet. </li><li> Oda sıcaklığında bir mikrosantrifüj içinde tam hızda 10 dakika boyunca dönerler. Sil süpernatant. </li><li> 10 dakika boyunca hava kuru topak. </li><li> Iki kez damıtılmış 100 ul içinde süspanse pelet daha önce H 2 O otoklava </li><li> Kullanılana kadar -80 ° C &#39;de bir sıvı sintilasyon sayacı içinde radyoaktivite, kısım radyo-etiketli prob İRE ve mağaza ölçmek. Donmuş madde şişeleri 3 haftaya kadar kullanılabilir. </li></ol> EMSA için yerli poliakrilamid jeli 5. N- <ol><li> 1,5 mm ara parçasını ve tarak yardımıyla jel (16 x 16 cm) birleştirin. </li><li> Tablo 3&#39;te gösterilen stok çözeltileri kullanmak,% 6 yerli poliakrilamid jel hazırlamak için 7.5 ml% 40 akrilamid karıştırın:. Bisakrilamid, 5x TBE 5 ml ve 37.5 mlçift ​​distile H 2 O </li><li> % 10 taze hazırlanmış amonyum persülfat (APS) ve 25 ul tetrametiletilenediamin (TEMED) 0.5 ml ekleyin. </li><li> Hemen jel akrilamid çözüm dökün ve polimerize edelim. Yaklaşık 30 dakika boyunca bekleyin. </li><li> , Jel ile elektroforez cihazı monte 0.5x TBE ile tanklarını doldurmak ve güç kaynağı ile bağlayın. </li></ol> 6. Elektroforetik Hareketlilik Kaydırma Deneyi <ol><li> 10 ul bir toplam hacim içinde hücreler ya da sitoplazmik lisiz tamponu (Tablo 1) dokudan elde edilen protein ekstresi, 25 ug seyreltin (düşük protein konsantrasyonları da kullanılabilmektedir). Buz üzerinde tutun. 25: 4 atıl IRP1 (% 2 nihai konsantrasyon) aktif hale getirmek için 2-merkaptoetanol (2-Me) ile seyreltildi: Gerekirse, 1 ve 1 ul ekleyin. </li><li> 95 ° çift distile H radyoaktif işaretli IRE probu sulandırmak 2 O 200.000 cpm / ul, ısı doğasını1 dakika için C ve en az 5 dakika boyunca oda sıcaklığında soğumaya. </li><li> Protein ekstresi Radyoetiketli IRE probu 1 ul ekleyerek bir EMSA reaksiyonu ayarlayın. </li><li> Oda sıcaklığında 20 dakika boyunca inkübe edin. </li><li> Reaksiyona 50 mg / ml heparin 1 ul ekle (prob 9 ile non-spesifik protein etkileşimlerini inhibe etmek için) ve 10 dakika daha inkübasyona devam etmektedir. -80 ° C de hazır heparin çözeltisi (50 mg / ml) ve mağaza alikosu. </li><li> Uzun radyo-etiketli problar (&gt; 60 nükleotid) kullanıldığında, 1 ul RNaz T1 (1 U / ul) ekleyin ve oda sıcaklığında 10 dakika inkübe edilir sondasına bağlanma, spesifik olmayan protein azaltmak için, RNA / protein daha iyi ayrılmasına izin vermek üzere Elektroforez sırasında kompleksi. </li><li> Yükleme tamponu (% 80 gliserol + bromofenol mavi) 3 ul ekleyin, karıştırın ve% 6 denatüre edici olmayan poliakrilamid jeli üzerinde yükü. </li><li> 130 V (5 V / cm) 60 dakika süre ile jel çalıştırın. </li><li> Transfer büyük bir filtre kağıdı ve kuru üzerine jel. </li><li> Bir film ortaya çıkarmak ve otoradyografi gelişir. Uygulama süresi 1 saat (ya da daha az) den O / N &#39;ye kadar çıkabilir. </li></ol>

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The authors declare that they have no competing financial interests.

Bu yazıda, IRP1 ve IRP2 ve IRE-bağlama faaliyetleri incelemek için geliştirilmiş bir protokol açıklar, ve biz temsilcisi verileri gösterir. Farklı problar kullanılarak, bu protokol, aynı zamanda, diğer RNA bağlama proteinlerinin çalışma için ayarlanabilir. Kritik bir safha, prob boyutu. Tam bağlanma yeri bilinmediğinde ortak uzun probların kullanımı, serbest RNA daha farklı geçiş olmayan RNA / protein kompleksleri ile sonuçlanabilir. Bu durumda, bu RNAz T1 (adım 6.6) ile muame...

EMSA başlangıçta, hedef DNA dizileri 1,2 DNA-bağlayıcı proteinler arasındaki ilişkiyi incelemek için geliştirilmiştir. ilkesi, bu makalenin odak noktası RNA / protein etkileşimleri 3 için benzer. Kısaca, RNA, negatif yüklü ve poliakrilamid (ya da agaroz) jelleri içinde denatüre edici olmayan elektroforez sırasında anoda doğru hareket edecektir. Jel içinde göç yüküne orantılı olan RNA, büyüklüğüne bağlıdır. Belirli RNA bir proteinin bağlanması, serbest RNA ile karşılaştırıldığında karmaşık göç eder yavaş hareketliliğinin değiştirir ve. Bu molekül kütlesinde bir artışın, aynı zamanda yük ve muhtemelen konformasyonda değişikler kaynaklanmaktadır. Prob olarak etiketlenmiş RNA kullanımı &quot;jel geriliği&quot; veya &quot;bandshift&quot; kolay izlenmesine olanak sağlar. 32 P-etiketli RNA prob kullanımı çok yaygındır ve yüksek hassasiyeti sunar. RNA / protein kompleksleri ve serbest RNA geçiş tespit edildiğindeOtoradyografi ile. Dezavantajları 32 P (14.29 gün), nedeniyle radyoliz prob kalitesinin kademeli bozulma kısa yarı ömrü vardır, bir radyoaktivite lisansı ve radyoaktivite çalışmaları için altyapı ve potansiyel biyogüvenlik kaygıları gereksinimi. Bu nedenle, bir RNA probu etiketlenmesine alternatif izotopik olmayan yöntemler, flüoresan veya kimyasal olarak ışık veren görüntüleme 4,5 radarından olanak florofor ya da biyotin ile, örneğin, geliştirilmiştir. Bu yöntemlerin Sınırlamalar yüksek maliyet ve izotopik etiketleme ile karşılaştırıldığında genellikle azalır duyarlılık ve RNA / protein etkileşimi ile müdahale olmayan izotop etiket potansiyeli vardır. Denatüre edici olmayan poliakrilamid jelleri en EMSA uygulama için uygun olan ve yaygın olarak kullanılmaktadır. Vesileyle, agaroz jelleri büyük kompleksleri analizi için bir alternatif oluşturabilir. EMSA büyük avantajı basitlik, duyarlılık ve sağlamlığı birleştiren 4 olduğunu &lt;/ Sup&gt;. Deney birkaç saat içinde tamamlanabilir ve karmaşık bir cihaz gerektirmez. RNA / protein etkileşimleri 0.1 nM veya daha az gibi düşük konsantrasyonlarda EMSA ile tespit edilebilir ve geniş bir bağlanma koşulları aralığında (pH 4,0-9,5, tek değerli bir tuzu konsantrasyonu 1-300 mM ve sıcaklık 0-60 ° C). RNA / protein kompleksi oluşumu, filtre bağlama deneyi ile incelenebilir. Serbest bir RNA probu 6 geçerken bu, bir nitroselüloz filtresi RNA / protein kompleksleri tutma dayalı bir basit, hızlı ve ucuz bir yöntemdir. EMSA ile karşılaştırıldığında, bu RNA sistemi birden çok bağlayıcı siteler, veya ham ekstrakt aynı sitede prob bağlanan birden fazla RNA bağlayıcı proteinler içermesi durumunda sınırlıdır. Birden fazla RNA / protein etkileşimleri filtre bağlayıcı tahliliyle tespit kaçış olsa da, onlar kolayca EMSA tarafından görüntülenmiştir olabilir. Bazı durumlarda, görselleştirme arifesidirn, mümkün olduğunca, jeli üzerinde daha fazla geriliği elde EMSA reaksiyonuna, RNA bağlanan proteinlerden biri karşı bir antikor eklenerek birlikte geçirmek (örneğin, insan IRP1 / İRE ve IRP2 / İRE kompleksleri) iki RNA / protein kompleksleri ( &quot;süperkayma&quot;) 7. EMSA yaygın demir metabolizması 8-10 transkripsiyon sonrası düzenleyiciler olan IRP1 ve IRP2, incelemek için kullanılır olmuştur. Onlar birçok mRNA 11 UTRs içinde İres, filogenetik korunmuş firkete yapıları bağlanarak çalışır. IRES ilk ferritin 12 ve transferrin reseptörü 1 (TfR1) 13, sırasıyla, demir depolanması ve alınması, proteinlerini kodlayan mRNA keşfedilmiştir. Daha sonra, İres eritroid özel aminolevulinate sentaz (ALAS2) 14, mitokondriyal akonitaz 15, ferroportin 16, iki değerlikli metal taşıyıcı 1 (DMT1) 17, hipoksi indüklenebilir faktör 2 bulundu <em&gt; α (HIF2α) 18, ve diğer mRNA&#39;lar 19-21. UTR&#39;de &#39;TfR1 mRNA onun 3 birden İres içeriyor ise, UTR&#39;de&#39; prototip H ve L-ferritin mRNAlar onların 5 bir IRE&#39;yi içerirler. IRE / IRP etkileşimleri özellikle sterik 43s ribozomal alt birim olan ilişkisini bloke ederek ferritin mRNA çeviri inhibe; Ayrıca, bu endonükleolitik klivaj karşı TfR1 mRNA&#39;nın stabilize eder. IRP1 ve IRP2 payı geniş sekans benzerliği ve demir-aç hücrelerde yüksek İRE bağlayıcı aktivitesi sergiler. IRP2 proteozomal bozulmaya uğrar ise demirin bol olduğu hücrelerde, IRP1 onun İRE bağlama aktivitesi pahasına sitosolik akonitaz dönüştürür bir küban Fe-S, küme bir araya getirmektedir. Bu nedenle, İRE / IRP etkileşimi hücre demir durumuna bağlıdır, fakat aynı zamanda, H2O 2, nitrik oksit (NO) veya hipoksi gibi diğer sinyalleri tarafından düzenlenmektedir. Burada, biz E ham hücre ve doku ekstrelerinden IRE-bağlama aktivitesinin değerlendirilmesi için protokol tarifMSA. Bu İRE dizisi aslında tarafından alt baş T7 RNA polimerazı site sens yönlenmede katılmasının bir plasmid DNA şablonu (I-12.CAT), in vitro transkripsiyonu ile üretilen bir 32 P-etiketli lH-ferritin İRE sonda kullanılan tavlanmış, sentetik oligonükleotidlerin 22 klonlanması.

<table border="0" cellpadding="0" cellspacing="0" width="430">
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			<td>BIORAD</td>
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			<td>Thermoscientific</td>
			<td>EPO111</td>
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			<td>Invitrogen</td>
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			<td align="right">141349</td>
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			<td height="17" style="height:17px;">heparin</td>
			<td>SIGMA</td>
			<td>H0777</td>
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			<td height="17" style="height:17px;">Rnase T1</td>
			<td>Thermoscientific</td>
			<td>EN0541</td>
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			<td height="17" style="height:17px;"></td>
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			<td height="17" style="height:17px;">Name of the Equipments&#160;</td>
			<td>Company</td>
			<td>Catalog Number&#160;</td>
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			<td height="17" style="height:17px;">Tissue Ruptor</td>
			<td>Qiagen</td>
			<td align="right">9001271</td>
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			<td>Beckman Coulter</td>
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			<td>BIORAD</td>
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			<td height="17" style="height:17px;">20 wells combs 1.5mm thick</td>
			<td>BIORAD</td>
			<td>165-1868</td>
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			<td height="17" style="height:17px;">1.5 mm spacers</td>
			<td>BIORAD</td>
			<td>165-1849</td>
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			<td height="17" style="height:17px;">PowerPac</td>
			<td>BIORAD</td>
			<td>164-5070</td>
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electrophoretic mobility shift assay (emsa) for the study of rna-protein interactions: the ire/irp example

RNA / protein etkileşimleri transkripsiyon sonrası düzenleme yolları için kritik öneme sahiptir. En iyi karakterize edilmiş olan sitosolik, RNA bağlanan proteinler arasında demir düzenleyici proteinler, IRP1 ve IRP2 bulunmaktadır. Onlar böylece mRNA&#39;ları çeviri veya stabilitesini kontrol, çeşitli hedef mRNA&#39;larının çevrilmemiş bölgelerde (UTRs) içinde duyarlı elemanları (İres) demir bağlama. IRE / IRP etkileşimleri yaygın EMSA tarafından incelenmiştir. Burada, biz de diğer RNA bağlayıcı proteinlerin aktivitesini değerlendirmek için jeneralize olabilir IRP1 ve IRP2 ve IRE-bağlanma aktivitesi, analiz için EMSA protokol açıklar. Bir RNA bağlayıcı protein ya da bu proteinin bir saflaştırılmış preparatı ihtiva eden bir ham protein lisatı, kompleks oluşumunu mümkün kılan, 32P-işaretli RNA-uydusuyla fazlası ile inkübe edilir. Heparin bağlayıcı prob, spesifik olmayan protein önlemek için ilave edilir. Daha sonra karışım, bir poliakrilamit jeli üzerinde denatüre elektroforezi ile analiz edilir. ücretsiz probhızlı göç ederken RNA / protein kompleksi sergiler engelli hareketlilik; dolayısıyla, prosedür de &quot;jel retardasyon&quot; veya &quot;bandshift&quot; tahlil denir. Elektroforezin tamamlanmasından sonra, jel kurutulur ve RNA / protein kompleksleri, hem de serbest prob, otoradyografi ile takip edilmiştir. Protokolün genel amacı algılamak ve IRE / IRP ve diğer RNA / protein etkileşimleri ölçümüdür. Ayrıca, EMSA da araştırılmaktadır RNA / protein etkileşiminin özelliğini, bağlanma afinitesini ve stokiyometri belirlemek için kullanılabilir.

Bölüm 3 ve protokol 4&#39;te tarif edildiği gibi bir radyo-etiketli prob İRE, elde edilmiştir. prob dizisi 5&#39;-GGGCGAAUUC GAGCUCGGUA CCCGGGGAUC CUG Cı UUCAA Cı AGUGC UUGGA CGGAUCCU-3 &#39;idi; kalın nükleotidler kritik IRE özellikleri bir eşleşmemiş C kalıntısı ve döngü, temsil eder. prob özel radyoaktivite 4.5 x 10 9 cpm / RNA ug idi. Aktivitesi İRE bağlayıcı demir sıçramaların etkilerini değerlendirmek için, sıça...

Watch this Scientific Journal Video about Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example at JoVE.com

Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Burada RNA / protein etkileşimleri analiz etmek için bir protokol mevcut. elektroforetik hareketlilik kayma deneyi (EMSA), yerli jel elektroforez sırasında RNA / protein kompleksleri ve serbest RNA ayırıcı geçiş dayanmaktadır. Bir radyo-etiketli bir RNA probu kullanarak, RNA / protein kompleksleri otoradyografi ile görselleştirilebilir.

IRE / IRP Örnek: RNA-protein Etkileşimleri Çalışmaları elektroforetik hareketlilik Shift Testi (EMSA)

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron ...

electrophoretic-mobility-shift-assay-emsa-for-study-rna-protein

Research

JoVE Journal

Biology

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

53.2K Görüntüleme.  Jewish General Hospital.  Burada RNA / protein etkileşimleri analiz etmek için bir protokol mevcut. elektroforetik hareketlilik kayma deneyi (EMSA), yerli jel elektroforez sırasında RNA / protein kompleksleri ve serbest RNA ayırıcı geçiş dayanmaktadır. Bir radyo-etiketli bir RNA probu kullanarak, RNA / protein kompleksleri otoradyografi ile görselleştirilebilir. 

Video: Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Kertenkeleler Hareket Hız ve Yön Visual Hassasiyet Test

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Biyoloji

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

Anjiyogenez Çalışmaları için optimize edilmiş Fibrin Jel Boncuk Testi

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Bilgisayar oluşturuldu Hayvan Modeli Uyaranlar

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

KRİYOPREZERVE Numune Kurtarma için Do-It-Yourself Aygıt yanlışlıkla Kriyojenik Depolama Tankları içine Dropped

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

Protein-Protein Etkileşimi Okumak Manyetik Boncuk Kullanarak Bir Sıvı Faz Affinity Yakalama Testi: Poliovirüs-Nanobody Örnek

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

EMBL de MultiBac Protein Complex Üretim Platformu

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Fonksiyon Çalışma, Etkileşimler, ve Post-translasyonel değişiklikler için tomurcuklanan Maya Protein Ekstraksiyonu ve Saflaştırılması

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

Protein-DNA Etkileşiminin Çalışmaları Tek molekül Manipülasyon ve Görüntüleme birleştiren

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Arabidopsis Protein-DNA Etkileşimleri Tespiti için Kromatin Immunoprecipitation Deneyi<em&gt; İn Vivo</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

Elektroforetik Hareketlilik Assay (EMSA) ve Shift kullanarak Fonksiyonel Olmayan kodlama Genetik Türevleri için tarama DNA afinite Yağış Deneyi (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical ...