This work was supported by FIRB-Merit grants RBNE08YFN3_005 and RBNE08HWLZ_012, and the Italian Ministry of Economy and Finance to the CNR for the Project &#8220;FaReBio di Qualit&#224;&#8221;.

1. siRNA çözümleri hazırlayın <ol><li> (50 uM son konsantrasyon siRNA) RNaz içermeyen su, 100 ul BRCA2 siRNA kurutuldu pelet 5 karıştırılmış (CTRL) nmol ve 5 nmol yeniden süspanse edin. Bu siRNA stok ve 10 ul alikotları -20 ° C&#39;de saklanmalıdır. </li><li> SiRNA stoktan 10 ul bir kısım alın ve 10 uM siRNA çalışma çözeltisi elde etmek için RNaz içermeyen su içinde 40 ul ekle. Bu seyreltme, kullanılıncaya kadar -20 ° C&#39;de saklanmalıdır. NOT: siRNA çalışma çözüm / dondurma geçmesi 3 kat daha fazla çözülme olmamalıdır. </li></ol> İnsan epitelyal hücre hattı olduğunu 2. Tohum <ol><li> 37 ° C&#39;de,% 5 CO2 nemlendirilmiş inkübatör içinde doku kültürü plakaları içinde hücre mono tabakaları olarak büyüyen insan epitelyal hücre hatları büyüme ortamı çıkarın. </li><li> Bir kez RT PBS ile plaka hücreleri (100 mm tabak için 10 ml PBS) yıkayın. </li><li> Plaka hücreleri ayırmak için,% 0.25 tripsin ve 2 ml / 0.53 mM EDTA çözeltisi ekleme ve hücre tipine bağlı olarak 3-5 dakika inkübe edin. </li><li> Müstakil hücreleri toplamak önce,% 10 fetal sığır serumu içeren tam büyüme ortamı 2 ml eklenerek tripsin nötralize eder. </li><li> 15 ml&#39;lik bir tüpe hücreleri aktarın. </li><li> Sallanan kepçeli rotor içinde 4 ° C&#39;de 3 dakika boyunca 320-330 xg&#39;de hücreleri santrifüjleyin. </li><li> Antibiyotikler-içermeyen ortamdan 5 ml hücre pelletleri yeniden süspanse edin. </li><li> Bir Bürker odasında mikroskop altında ya da imalatçının talimatlarına uygun olarak, otomatik bir sayma sistemi kullanarak hücreleri sayın. </li><li> 6-delikli plakalardaki hücreler 24 saat (de 0.5-1 x 10 her biri için ortam 2 ml 6 hücre) sonra yaklaşık% 80 konfluent olarak tohumu. Optimal hücre sayısı özel hücre çizgileri, büyüme oranına bağlı olarak değişebilir tohumlanır edilmesi. NOT: antibiyotikler olumsuz transfe verimliliği engel olacak çünkü bu noktada ortamda antibiyotik katmayınction. Bu BRCA2 siRNA transfeksiyon, yüksek verim elde etmek için, düşük bir kültür geçitlerin (&lt;10) en hücreleri olması çok önemlidir. </li></ol> SiRNA ile 3. Transfect hücreleri <ol><li> Yirmi dört saat sonra, hücreler kaplama sonrası, transfeksiyon ilk tur devam edin. <ol><li> Indirgenmiş serum ortamı 130 ul lipid bazlı 10 siRNA belirli transfeksiyon reaktif 6 ul sulandırmak. </li><li> Indirgenmiş serum ortamı 130 ul 10 uM siRNA (30 pmol) 3 ul sulandırmak. </li><li> Seyreltildi transfeksiyon reaktifi ile seyreltildi siRNA birleştirin ve oda sıcaklığında 5-10 dakika boyunca inkübe edin. </li><li> İnkübasyon sırasında, hücrelerden orta kaldırmak ve taze ortam (antibiyotiksiz), 37 ° C&#39;de önceden ısıtılmış 2 ml ekleyin. </li><li> Hücrelere siRNA transfeksiyon reaktifi komplekslerinin 250 ul (bu miktar, 6 gözlü bir plakanın tek oyuk için) ekleyin. </li><li> 37 ° C&#39;de, nemlendirilmiş bir kuluçka makinesi içinde hücreleri inkübe76;% 5 CO2 C. </li></ol></li><li> 24 saat sonra, adım 3.1.2 şu değişiklikler 3.1.1-3.1.5 adımları tekrarlayarak transfeksiyon ikinci tur devam: düşük serum ortamı 130 ul 10 uM siRNA (50 pmol), 5 ul seyreltilir. <ol><li> Analizden önce 37 ° C&#39;de 1-2 gün süreyle inkübe hücreleri. NOT: transfeksiyonları ve ikinci turda yüksek bir siRNA / transfeksiyon reaktif oranına kullanımının iki tur BRCA2 sessizliğini yüksek verim elde etmek için gereklidir. </li></ol></li></ol> Immunoblotting analizi için 4. Hücreleri, <ol><li> Iyonik-olmayan, denatüre edici olmayan deterjan oktilfenoksipolietokisetanol% 1, 5 mM sodyum pirofosfat, 1 mM sodyum ortovanadat, 10 mM sodyum flüorür, 2 mM fenilmetilsülfonil fluorür, 10 ug içeren PBS (pH 7.4), taze liziz tamponu / ml löpeptin, 10 | ig / ml aprotinin. Buz üzerinde tutun. </li><li> Hücreler ve w orta çıkarınlevhanın soğuk PBS (2 ml / oyuk) ile bir kez bunları kül. Herhangi bir PBS taşınmasını önlemek için, tamamen plaka çıkarın. </li><li> Her bir oyuğa liziz tamponu içinde 200 ul ekle. NOT: hücreler, diğer analizler için de kullanılabilir hücreleri trypsinize aşama 2,1-2,6 tarif edildiği gibi ve uygulamaya göre, uygun tampon / ortam içinde hücreler tekrar süspansiyon gerekir ise kullanılır. </li><li> Yavaşça eşit lizis tamponu dağıtma ve bir döndürücüde 4 ° C de 15 dakika inkübe etmek plaka döndürün. </li><li> Bir hücre kazıyıcı kullanılarak buz üzerinde, bir mikrosantrifüj tüpü içinde, hücre lizat toplayın. </li><li> 4 ° C&#39;de 25 dakika boyunca 17.900 x g&#39;de santrifüje ve yeni bir tüp içinde supernatant toplamak. </li><li> Üreticinin talimatlarına göre bir ticari olarak temin edilebilen protein deneyi kullanılarak protein konsantrasyonu ölçümü. </li></ol> 5. BRCA2 immunoblotting analizi <ol><li> 20x Tris-Asetat SDS çalışan b 50 ml seyreltilmesi ile çalışan tampon hazırlayınUffer (50 mM Trişin, 50 mM Tris baz,% 0.1 SDS, pH 8.24) damıtılmış su 950 ml. </li><li> Aşağıda örnek hazırlayın: 20 nihai bir hacme kadar 15 toplam hücre lizatı ug, 4x numune tamponu içinde 5 ul pH 8.4 lityum dodesil sülfat içeren, 10x Örnek indirgeyici madde (0.5 M DTT) 2 ul ve lizis tampon eklemek ul. NOT: Bu protokol, (5 ug kadar), aynı zamanda, toplam proteinlerin daha düşük bir miktarda kullanarak, normal hücre çizgilerinde BRCA2 saptanmasını sağlar. </li><li> 10 dakika boyunca 55 ° C&#39;de numune denatüre. NOT: 55 ° C kullanmak çok önemlidir. BRCA2 proteini ısıya 11 olduğu için, daha yüksek sıcaklıklar, denatüre edici tam boy (390 kDa), BRCA2 proteininin tutarlı bir azalmaya neden olur. </li><li> Üreticinin talimatlarına göre elektroforetik bölmeyi ayarlayın. </li><li> Örnekler ve precasted jeli üzerinde yüksek molekül ağırlıklı ön-lekeli protein standardı 10 ul (Tris-Asetat% 3-8) yük ve ab 120 V çalıştırmak2 saat 12. 55 kDa mavi işaretleyici precasted jel ayrılmadan önce elektroforetik akışını durdurur. </li><li> 20x Transfer tamponu, 50 ml% 100 metanol, 100 ml ve 850 ml su ile birlikte Transfer tamponu hazırlayın. </li><li> 5 dakika boyunca% 100 metanol içinde ıslatarak PVDF membranı (0.45 mikron gözenek büyüklüğü) aktive damıtılmış su ile yıkayın ve en az 5 dakika için transfer tamponu içinde dengeye. Kullanılana kadar 4 ° C &#39;de transfer tampon maddesi transfer cihazının sünger bekletin. </li><li> Elektroforetik çalışma bittiğinde, aşağıdaki sırayla (aşağıdan yukarıya) transfer yığınında sandviç monte: transfer tamponu doymuş üç süngerler, tek 3MM sınıfı kağıt levha transfer tamponu, jel, aktif PVDF membran, tek 3mm doymuş Transfer tamponunda doymuş sınıf kağıt levha, üç süngerler transfer tamponu 13,14 doymuş. 4 ° C&#39;de gece boyunca mA 4 ° C ya da 180 ° C&#39;de 4 saat 350 mA&#39;da cihazı ve aktarım proteinleri içine yığın yerleştirin. NOT: yüksek molekül ağırlıklı proteinlerin tam transferini sağlamak üzere 4 saat veya gece için (üretici ve çoğu aktarım protokolleri tarafından önerilen) olmak BRCA2 çok büyük bir protein o 1 saat transfer süresini uzatmak için çok önemlidir. Buna ek olarak, uzun bir aktarım zaman, bu aşırı ısınmayı en aza indirmek ve sandviç Sökme kolaylaştırmak için 4 ° C &#39;de soğuk bir odada blotting işleminin gerçekleştirilmesi için çok önemlidir. Yarı kuru transfer sistemleri büyük proteinlerin aktarımı için iyi değildir. </li><li> Aktarma işleminden sonra (30 ul anti-BRCA2 tavşan poliklonal antikoru ile 4 ° C&#39;de bir gece boyunca% 5 yağsız kuru süt ve probu içeren TBS-Tween% 0.25 içinde oda sıcaklığında 1 saat (TBS-T) için bloke TBS ile zarın yıkanması [: 300 (h / h) sulandırma 1] 9 ml TBS-T +% 5 yağsız kuru süt içinde seyreltilmiş 200 ng / ml). </li><li> Daha sonra TBS 10 ml seyreltilmiş bayır turpu peroksidazı ile konjuge edilmiş anti-tavşan ikincil antikoru ile 1 ul, 1 saat boyunca filtre inkübe, TBS-T ile, membran üç defa (10 dk) yıkayın-T +% 5 yağsız kuru süt. Bundan sonra, TBS-T ile membranı üç defa (10 dk) yıkayın. </li><li> Gelişmiş kemilüminesans (ECL) Western Blot saptama reaktif maddesi kullanılarak, üretici talimatlarına göre BRCA2 proteini ortaya çıkarmak için ECL tarafından immunodeteksiyon gerçekleştirin. Yüksek çözünürlüklü ECL filmlerinde immunochemiluminescent bantları gözünüzde canlandırın. </li><li> 10 anti-tubulin antikoru 1000 (10 ul: β-tubulin bir monoklonal antikor kullanılarak 1 seyreltilmiş göre yükleme kontrolü olarak, aynı filtre üzerinde, β-tubulin (55 kDa) gibi bir ev bakıcısı geni için bir antikor ile immunodeteksiyon gerçekleştirme oda sıcaklığında 1 saat ml TBS-T +% 5 yağsız kuru süt). Yaban turpu peroksidazı ile konjuge edilmiş anti-fare yerine anti-tavşan sekonder antikor kullanılması haricinde, yıkama ve BRCA2 (5,10-5,11) için tarif edildiği gibi sekonder antikoru inkübe edin. </li><li> (Immunochemiluminescent bantları ile etkilendim filmlerin dijital görüntü elde ve özel görüntü analiz yazılımı tarafından bant yoğunluğunu analiz, örneğin., ImageJ). </li></ol>

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The authors have nothing to disclose.

Kadın ve erkek meme kanseri, yumurtalık kanseri, prostat kanseri, pankreas kanseri ve melanom 3,4 dahil olmak üzere birçok kanser türleri, risk artışına BRCA2 geni kurşun germline mutasyonlar, çok sayıda çalışma biyolojik işlevini anlamak için yapılmıştır Çünkü BRCA2 protein. Bu çalışmaların çoğu nedeniyle BRCA2 gibi dev bir protein analiz teknik zorluklar genetik temelli çoğunlukla. BRCA2 protein ekspresyonu susturma ve analiz edilmesi için burada tarif edilen metot, ...

BRCA2 (Meme Kanseri yatkınlık geni-2) gen rekombinaz enzimi Rad51 1 işlevini düzenleyen DNA çift iplikli sonları onarımında önemli bir rol oynayan bir tümör baskılayıcı protein kodlayan. BRCA2 aynı zamanda transkripsiyonun modülasyonu ve hücre döngüsü kontrol 2 implike edilmiştir. BRCA2 geninin germline mutasyonlar için diğer kanser türleri 3,4 otozomal dominant erkeklerde meme ve kadınlarda yumurtalık kanseri ve prostat kanseri olan yatkınlığı, hem de yatkınlığı neden olur. Ancak, bastırmak ya da DNA hasarına neden olan 5-7 kemoterapötik ajanların kanser hücrelerinin daha hassas hale getirebilir BRCA2 fonksiyonunu azaltmak kanser, BRCA2 mutasyonları gelişmekte riski rağmen. Sporadik kanserler o BRCA2 protein düşündüren, BRCA2 proteininin azaltılmış düzeylerinin bazı kanser türlerinde tespit edilmiştir rağmen BRCA2 mutasyonları (&lt;% 3) düşük bir oran sergilemektedirolmayan mutasyon bağımlı mekanizmalar 7,8 ile sporadik kanserlerde tümör oluşumu sırasında kaybolmuş olabilir. Bu nedenle, tam Kanser biyolojisinde bağlamında gibi diğer biyolojik ortamlarda BRCA2 fonksiyonlarını anlamak önemlidir. Memeli hücrelerinde gen yeni işlevleri belirlemek için kullanılan güçlü bir araçtır ifadesini susturma etmektir. Bir örnek olarak, normal epitel hücrelerinin çeşitli susturma BRCA2 sentezleme en son anoikis direnci, kanser hücresi invaziv ve metastatik yeteneği 9 edinilmesi sırasında önemli bir aşama düzenleyici olarak BRCA2 yeni bir fonksiyon belirlenmesine yol açmıştır. Gen susması hücrelerin küçük müdahale (si) RNA ya da özel bir geni hedef alan kısa bir saç tokası (sh) RNA molekülleri ya sokulması ile elde edilebilir. Yüksek siRNA gücü ve kullanım kolaylığı kritik biyolojik süreçler içine yeni anlayışlar kazanma amaçlı deneyler susturmak için onu tercihli aracı yapmak birnd yeni tedavi hedefleri tespit etmek. Ancak, gen ekspresyonunun etkili bir yok etme tüm genler kolaylıkla elde olmayabilir ve hücre türüne bağımlı olarak oldukça değişken olabilir. Hücre içi protein seviyelerinde bir azalma araştırılmaktadır en uygun fenotip olduğu için, gen ürününün analizi immün gen susmasını ölçmek için son derece önemlidir. Bu bağlamda, BRCA2 proteininin bir başka meydan okuma sunuyor: Büyük bir protein (yaklaşık 390 kDa) olan, teknik zorluklar immunoblotting dahil olmak üzere geleneksel biyokimyasal analiz için mevcut. Biz burada insan epitel hücre hatlarında BRCA2 verimli susturulması için ve analiz immün demonte BRCA2 proteininin hızlı ve başarılı tespiti için optimize edilmiş bir protokol rapor.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>BRCA2 siRNA</td>
			<td>Dharmacon</td>
			<td>L-003462-00</td>
			<td>SMARTpool: ON-TARGETplus BRCA2 siRNA</td>
		</tr>
		<tr>
			<td>Ctrl siRNA</td>
			<td>Dharmacon</td>
			<td>&#160;D-001810-10-05</td>
			<td>ON-TARGETplus Non-targeting Pool</td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMAX Reagent&#160;</td>
			<td>Life Technologies</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/13778075">13778075</a></td>
			<td>Transfection Reagent</td>
		</tr>
		<tr>
			<td>OPTI-MEM I Reduced Serum Medium</td>
			<td>Life Technologies</td>
			<td>31985-070</td>
			<td>Medium for transfection procedure</td>
		</tr>
		<tr>
			<td>NuPAGE Tris-Acetate 3-8% 1,5 mm Gel precast</td>
			<td>Life Technologies</td>
			<td>EA0378</td>
			<td>Gel for protein electrophoresis</td>
		</tr>
		<tr>
			<td>NuPAGE LDS Sample buffer&#160;</td>
			<td>Life Technologies</td>
			<td>NP0007</td>
			<td>Reagent for protein electrophoresis</td>
		</tr>
		<tr>
			<td>NuPAGE Sample Reducing agent (10x)</td>
			<td>Life Technologies</td>
			<td>NP0004</td>
			<td>Reagent for protein electrophoresis</td>
		</tr>
		<tr>
			<td>NuPAGE Antioxidant</td>
			<td>Life Technologies</td>
			<td>NP0005</td>
			<td>Reagent for protein electrophoresis</td>
		</tr>
		<tr>
			<td>HiMark Pre-stained protein standard</td>
			<td>Life Technologies</td>
			<td>LC5699</td>
			<td>Prestained marker for gel electrophoresis</td>
		</tr>
		<tr>
			<td>XCell SureLoc Mini-Cell&#160; System</td>
			<td>Life Technologies</td>
			<td>EI0002</td>
			<td>Equipment for protein electrophoresis/transfer</td>
		</tr>
		<tr>
			<td>NuPAGE Tris-Acetate SDS Running Buffer</td>
			<td>Life Technologies</td>
			<td>LA0041</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>NuPAGE Transfer Buffer</td>
			<td>Life Technologies</td>
			<td>NP0006-1</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>BRCA2 H-300 rabbit polyclonal antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-8326</td>
			<td>1:300 dilution</td>
		</tr>
		<tr>
			<td>Beta-tubulin monoclonal antibody</td>
			<td>Sigma Aldrich</td>
			<td>T4026-100UL</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Skim Milk powder</td>
			<td>Sigma Aldrich</td>
			<td>70166</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma Aldrich</td>
			<td>P1379</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico/Femto Chemiluminescent Substrate</td>
			<td>Pierce Thermo Scientific</td>
			<td>34080/34096</td>
			<td>Chemiluminescence system</td>
		</tr>
		<tr>
			<td>PNT1A cells</td>
			<td>SIGMA Aldrich (for ECACC)</td>
			<td>95012614</td>
			<td>PNT1A human, normal prostate epithelium immortalized with SV40</td>
		</tr>
		<tr>
			<td>Nthy cells</td>
			<td>SIGMA Aldrich (for ECACC)</td>
			<td>90011609</td>
			<td>Nthy-ori 3-1 Cell Line human, thyroid follicular epithelial cells</td>
		</tr>
		<tr>
			<td>Cell Proliferation Kit I (MTT)</td>
			<td>Roche</td>
			<td>11465007001</td>
			<td>Kit for cell proliferation assays</td>
		</tr>
	</tbody>
</table>

silencing of brca2 to identify novel brca2-regulated biological functions in cultured human cells

Tümör baskılayıcı protein BRCA2 ve konvansiyonel biyokimyasal analizler tarafından tespit susturma insan BRCA2 proteininin (yaklaşık 390 kDa) büyüklüğü sayesinde büyük bir teknik sorun oluşturmaktadır. Biz insan epitel hücre hatlarında sırasıyla endojen BRCA2 proteini, insan BRCA2 susturmak ve tespit etmek için standart siRNA transfeksiyon ve immunoblotting protokolleri değişiklikler rapor. Anahtar adımlar transfeksiyon reaktif oranı yüksek siRNA ve aynı deney içinde siRNA transfeksiyon birbirini takip eden iki mermi bulunmaktadır. Anti-BRCA2 antikorlar ile analiz immün tarafından değerlendirilecek sürekli olarak insan BRCA2 geninin fazla% 70 susturulması elde standart protokole bu ve diğer değişiklikler kullanma. Buna ek olarak, bunun yerine klasik 70-100 ° C ve immunoblotting prosedür diğer teknik iyileştirmeler 55 ° C&#39;de hücre lizatlarının denatürasyon sağlam BRCA2 proteininin tespiti da WH izinbaşlangıç ​​malzemesinin çok düşük miktarlarda en mevcuttur ya da BRCA2 protein ekspresyon seviyesi çok düşük olduğunda. Insan hücrelerinde BRCA2 etkin susturulması, tümör hücreleri de dahil olmak üzere sağlam BRCA2, hücrelerde BRCA2 fonksiyonunu bozmaya yeni moleküler yollar ve tümör patojenik BRCA2 mutasyonu etkilenebilir hücre fonksiyonlarını incelemek için değerli bir strateji vardır. Verimli susturma ve BRCA2 gibi diğer &#39;büyük&#39; ​​protein analizi için bu protokolün Adaptasyon kolayca ulaşılabilir olmalıdır.

Hücre fonksiyonları üzerine BRCA2 susturma etkisini incelemek üzere bir biyolojik / biyokimyasal tahlil geçmeden önce, ilk adım bir şifreli siRNA (siRNA olmayan hedefleme) BRCA2 siRNA ile yan yana (Şekil 1A kullanarak susturma özgünlüğünü doğrulamak için ). Bu BRCA2 susturma yüksek verim elde etmek için yüksek bir siRNA / transfeksiyon oranı ile siRNA transfeksiyon ikinci tur gerçekleştirmek için önemlidir. Gerçekten de, siRNA transfeksiyon tek aşama performa...

Watch this Scientific Journal Video about Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells at JoVE.com

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented.

Susturulması<em&gt; BRCA2</em&gt; Kültür İnsan hücrelerinde Roman BRCA2 düzenlenmiş Biyolojik Fonksiyonları Belirlemek İçin

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the ...

silencing-brca2-to-identify-novel-brca2-regulated-biological

Research

JoVE Journal

Biology

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

9.0K Görüntüleme.  National Research Council. Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented. 

Video: Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

İlköğretim Kültür Hipokampal Nöronlar Yoğun çekirdekli veziküller Canlı Görüntüleme

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

Biyoloji

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Ribozom Bağlı Doğan Polipeptitler İzolasyonu<em&gt; In vitro</em&gt; MRNA boyunca Translasyonel Duraklat Siteleri Belirleme

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

Gen İfadesi DNA etkileşimleri, Discover Transkripsiyonlu Regülatörleri ve Roman Anti-tümör Ajanlar tanımlayın: Protein Eğitim için Kantitatif Testi

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

Kültür Hücrelerinin retikulum Subdomain&#39;ler Görselleştirme

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

Yakalama Bileşik Kütle Spektrometre - Roman c-di-GMP Efektör Proteinler tanımlamak için güçlü bir araç

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Kültür Memeli Hücrelerde Biyolojik tanımında ubikitin ve Ubiquitin benzeri Sistemi Regülatörleri tanımak siRNA Eleme

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Zebra balığı Nephrotoxin Mikroenjeksiyon Akut Böbrek Yaralanması Model

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

Yüksek çözünürlüklü Time-lapse Görüntüleme ve Yaşayan İnsan Umbilikal Ven Endotelyal Hücrelerinde Mikrotubul Dinamiği Otomatik Analizi

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Dinamik Hücresel Protrüzyonlarda Protein Konsantrasyonunun Yazılım Destekli Takibi için Grafiksel Bir Kullanıcı Arayüzü

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

Glikoz Alımı Ölçümü ve İnsülin Uyarıcısına Yanıt<em&gt; In vitro</em&gt; Kültürlü İnsan Primer Miyotları

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...