Bu yayının önde gelen iş GA No 246550 altında Malaga Üniversitesi ve Avrupa Birliği 7. Çerçeve Programı (FP7/2007-2013) mali katkısıyla Marie Curie COFUND programı &quot;U-Mobility&quot;, fon aldı. Laboratuvarımızda çalışma VC NIH, USDA / Nifa, NSF, ozan ve BSF hibe tarafından desteklenmektedir

1.. Protein ekspresyonu <ol><li> Ekspresyon sistemi seçimi Sistemin, örneğin, vektörler ve spesifik bir model organizma / hücrede ilgi konusu proteinin ekspresyonu için en uygun vektör dağıtım yöntemi seçin. Bizim deney iyi hücre sayıda geçici transformasyonu ile elde edilir kolayca algılanabilir miktarlarda, test edilen proteinlerin ifadesini gerektirdiğini not edin. Bitkilerde, örneğin, bu dağıtım sistemi en iyi ekspresyon vektörleri Agrobacterium ikili plazmidler kullanılarak gerçekleştirilir. </li><li> Ikili sentezleme vektörlerinin yapılandırılması Bir epitop etiketine tek başına ya da translasyonel füzyon ya da bir ekspresyon vektörü içine ilgilenilen protein (ler) in kodlama sekans (lar) Clone. Kullanım olarak seçilen sentezleme sisteminde ve gen klonlama için kullanılan standart moleküler biyoloji işlemleri için uygun olan vektörleri. Agrobacterium-aracılı gen aktarımı için, Standa kullanılması da, ikili vektörler kullanmak ve bunları tanıtmakBitki dokularının aşılanması için daha sonra bu gibi bir EHA105 Agrobacterium suşu, içine rd protokolleri. </li><li> Bitki türlerinin seçimi Ilgilenilen protein (ler) olarak ifade edilecek olan bitki türleri seçin. Agrobacterium aracılı genetik transformasyona duyarlı herhangi bir bitki türü kullanılabilir iken, unutmayın, seçim bizim bitki türlerinin kolayca Agrobacterium&#39;a son derece duyarlı, yetiştirilen Nicotiana benthamiana, ve kolayca aşılanır büyük yaprakları vardır. </li><li> Bitki büyüme Uzun bir günün çevre kontrollü koşullarda (örneğin, büyüme odası) 130 (yani, 16 saat altında bir tencerede (20 cm x 20 cm x 20 cm) Pro-Mix BX ile bir tencerede 4 ila 6 hafta boyunca bir bitki büyütün 23 ° C&#39;de -150 μE m-2 s-1, ışık ve 20 ° C&#39;de 8 saat karanlık) ve% 40-65 bağıl nem. Zaman, üretici talimatlarına göre ticari olarak temin edilebilen ürünler ile 1.5.2 Gübre. Bitki yetişir kez seçmekAgrobacterium aşılama için 50 mm x 70 mm ya da daha büyük bir boyutu (uzunluk, bu ölçümler sapı kapsamamaktadır) ile terk eder. </li><li> Agrobacterium ile aşılama / Uygun antibiyotikler (örneğin, 100 mg desteklenmiş YEP ortam içinde 28 ° C&#39;de (% 1 pepton,% 1 maya özütü ve% 0.5 NaCl), gece boyunca adım 1.3 arasındaki test edilen protein (ler) ifade eden ikili yapıyı taşıyan Agrobacterium suşu büyümek L streptomisin ve pPZP RSC2-bazlı vektörler, 24-25) için 10 mg / L rifampisin. [10 mM MgCI2, 10 mM Mes (pH 5.6), 100 uM asetosiringon] sızma tamponu = 0.5 OD 600 ve oda sıcaklığında 2 saat boyunca inkübe yeniden süspansiyon haline 1.6.2 Santrifüj hücreler. 1 ml iğnesiz şırınga kullanılarak, bir yaprağın eksen dışı tarafına kültür sızın, bitkiye takılı durumdayken yapraklar, in situ aşılanır unutmayın. 1.6.3 infiltrasyonu sonra 16 saat 130-150 ve ışık rejimi altında 72 saat boyunca bitki büyümesi# 181; E, m-2 s-1 ışık 23 ° hasat öncesi 20 ° C ° C / 8 saat karanlık. </li><li> Proteozomal inhibitörlerinin uygulanması Test protein (ler) bir proteozomal yolu aracılığıyla bozulmuş olduğu görüşünü desteklemek için, azaltmak ve hatta bozulmasını engellemek gerekir, hangi spesifik proteozomal inhibitörleri MG132 kullanımı ve 28-29 Laktasistin. Dört saat (adım 2.1) hasat önce, 10 uM MG132 (Merck) ya da 10 uM laktasistin (Sigma-Aldrich) ya da Agrobacterium ile aşılanmış yaprak alanları sızmak ilgili çözücülerin, yani,% 0.1 DMSO ile mock-yaprak tedavisinde ya da damıtılmış su. </li></ol> 2. Hücresiz ekstrelerinin hazırlanması <ol><li> Yaprak hasat Yaprağın aşılanmış alanları, genellikle ca hasat. 200-400 taze ağırlığının, herbir mg, ve sıvı azot içinde ince bir toz haline öğütmek bunları. Alternatif olarak, herhangi boncuk-çırpıcı (örneğin, BioSpec) veya diş amalgamator (örneğin, TCK Advanced kullanarak, dokuları boncuk yendi<html> Doğrudan bozulma tamponunda Technologies) () Adım 2.2. </li><li> Protein ekstre etme Bozunma tampon 500 uL içine zemin dokusu yerleştirerek total protein ekstresi hazırlamak [50 mM Tris-HCL (pH 7.5), 100 mM NaCl, 10 mM MgCl2, 5 mM DTT, 5 mM adenozin 5&#39;-trifosfat ve 1x proteaz inhibitör kokteyli (Sigma-Aldrich)]. Proteaz inhibitörü kokteyl esas olarak serin, sistein, aspartik ve metaloproteazları etkiler ve 26S proteaz ile karışmaz unutmayın. 5 dakika boyunca 12,000 x g&#39;de iki ardışık santrifüj işlemi ile özü açıklığa kavuşturulacaktır. </li><li> Protein bozunma reaksiyon Mikrofüj tüpler ve zaman süreleri arttırmak için oda sıcaklığında inkübe etmek, özler, genellikle 20 uL&#39;lik eşit hacimlerde aktarın. Tipik haliyle, numune süresi sıfır, ve 5, 10, 15, 20 ve 30 dakika zaman noktalarında. SDS jel örnek tampon maddesi içinde kaynatma ile reaksiyonlar durdurun ve western blotting ile analiz. </li></ol>jove_title &quot;&gt; 3. imünoblotlama ile protein parçalanmasını algılama <ol><li> Jel elektroforezi 3.1.1 SDS-poliakrilamit jel elektroforezi ile protein örnekleri gidermek ve standart protokole 30&#39;a göre bir nitroselüloz zara giderilmiş proteinleri elektrotransfer. 3.1.2 Bradford yöntemi (Bio-Rad) kullanarak protein konsantrasyonunu belirleyin ve hat başına toplam proteinin 50-80 ug yükleyin. Tüm örnekler eşit yüklü olduğundan emin olun. Yükleme kontrol için, bu yaklaşık 50 kDa bir görece elektroforetik hareketlilik ile önemli bir bant olarak göç varsayımsal RUBISCO büyük zincirler olarak her yerde bulunan bir protein türleri, yoğunluklarını karşılaştırmak; bunlar Coomassie blue-lekeli jeller üzerinde veya Ponceau S-ya da tespit edilebilir Floresan SYPRO Ruby nitroselüloz membranlar-boyandı. </li><li> Bloke etme Oda sıcaklığında 1 saat boyunca TBST içinde% 5 yağsız süt (10 mM Tris-HCI, 140 mM NaCI,% 0.05 Tween 20, pH 7.4) ile bloke membran. </li&gt; <li> Primer antikor Üretici tarafından tavsiye edilen konsantrasyona TBST içinde% 1 yağsız süt birincil anti-antikor epitop seyreltin ve yumuşak çalkalama ile 4 ° C&#39;de, oda sıcaklığında 1 saat süre ile ya da gece boyunca bloke edilmiş membran ile inkübe edin. </li><li> Durulama Membran, 15 dakika boyunca bir kez 20 ml TBST içinde çalkalanır durulayın ve iki kez nazik çalkalama ile oda sıcaklığında 5 dakika karıştırıldı. </li><li> İkincil antikor Üretici tarafından tavsiye edildiği gibi TBST içinde% 1 yağsız süt olarak at turpu peroksidaz (HRP) ile konjüge edilmiş sekonder antikor (örneğin, bir anti-tavşan IgG) seyreltin ve yumuşak çalkalama ile oda sıcaklığında 1 st için membran ile inkübe edin. </li><li> Bulma Aşama 3.4 &#39;de tarif edildiği gibi yeniden membran durulayın. TBST içinde son bir kez çalkalandıktan sonra, en yaygın olarak, bir ECL kiti kullanılarak, bir HRP alt-tabaka ile kimyasal olarak ışık veren, ilgi konusu proteinleri görselleştirmek. </li></ol>

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Çıkar çatışması ilan etti.

Bu deney, bitki dokularında test edilen proteinlerin ekspresyonu dayanır, böylece, potansiyel proteozomal bozulması işlemi belli canlı dokular içinde daha önce ortaya çıkar. Sıfır örnek başlangıç ​​referans noktası olarak hizmet eden zaman, sadece ekstrelerinde ise, protein kararsız hale gelmelerine tahlil. Bu yüzden, bir hücre içermeyen tahlil olarak tanımlar. Bu deneyde başarısı için önemli bir yönü, test edilen protein (ler) üretilecek olan ifade vektör?...

Ubiquitin/26S proteazomu yolu diğerleri arasında transkripsiyonel regülasyonu, hücre döngüsü ilerlemesi ve sinyal iletimi, reseptör down-regülasyonu veya endositoz, 1-4 işler dahil olmak üzere çeşitli biyolojik reaksiyonlar, kontrolü için yaygın bir mekanizma olarak ortaya çıkmaktadır. Bu yol, hedef protein ilk ubikitin aktive edici enzim E1 için bir tiolester bağ üzerinden birleştirilmiş ve daha sonra ubikuitin konjugasyon enzim E2&#39;nin bir sistein amino asit kalıntısına transloke tortuları ubikuitin ile etiketlenir, son olarak, E2, E3 ligaz ubiquitin ile etkileşime , protein alt-tabakanın polyubiquitination ile sonuçlanır. Sonuç olarak, proteinler polyubiquitinated 26S proteazomu tarafından tanınan ve parçalanırlar. Bu mekanizmada, E3 enzim substratı belirtir ve ubiquitin/26S proteazom sistemi (UPS) en önemli düzenleyici bir bileşeni olarak işlev görür. E3 ligaz gibi halka alanı ligazlar olarak, ya da bir multisubunit SCF (S parçası olarak, bağımsız bir şekilde hareket edebilirBöyle F-box etki ligazlara olarak kp1/Cullin/F-box protein) kompleksi. SCF aracılı proteozomal degradasyon yollarının transkripsiyon, hücre döngüsü, sinyal transdüksiyonu 5-10 ve birçok diğer önemli hücresel fonksiyonların düzenlenmesinde rol oynamaktadır. Hücresel süreçlerin düzenlenmesinde bu kritik rolleri yanı sıra, UPS birçok bitki-patojen etkileşimleri merkezi sahne alıyor. Örneğin, artan kanıtlar Agrobacterium tumefaciens dahil olmak üzere birçok bitki patojenleri, enfeksiyon süreci 11 kolaylaştırmak için konakçı UPS güvenmek olduğunu göstermektedir. Agrobacterium doğal ana temsil eden bitkiler, neoplastik büyümeler ortaya çıkarır, ve aynı zamanda insan hücreleri 12,13 için mantar 1,2 dan, ökaryotlarda geniş bir yelpazede dönüştürebilir. Bu enfeksiyon esnasında, Agrobacterium konakçı hücre 12-13 bir DNA elemanı (T-DNA) ve çeşitli virülans (vir) proteinleri ihraç etmektedir. Bu proteinlerin biri VirF, bulunan ilk F-box proteindirBir prokaryotik genom 14 ile kodlanmış olması. SCF ubikuitin ligaz kompleksi, VirF ve onun fonksiyonel bir ev sahibi homolog VBF 15 bir parçası olarak, muhtemelen VirE2, ona eşlik eden bir bakteriyel ve konak proteinler istilacı bakteri T-DNA&#39;nın integraz kolaylaştırır UPS-aracılı protein yıkımı, Agrobacterium ile enfeksiyon kolaylaştırmak VIP1 ve sırasıyla 16,17. İlginçtir, VirF dahil olmak üzere birçok F-box proteinler, nedeniyle autoubiquitination aktivite 18,19 veya F-box proteinler yüzeylerde 20-23 olarak hizmet olabilecek diğer E3 ligazlara aracılık kendi proteoliz, özünde kararsız. F-box proteinler, diğer ligazlar ubikitin ve / veya alt-tabakalar arasında biyokimyasal aktivitelerini incelemek, bu proteozomal indirgenmesi için basit ve güvenilir bir deney kullanılması çok yararlı olacaktır. Burada, bir hücrede protein stabilitesini analiz etmek için böyle bir protokol açıklar-Free sistemi. Bu deneyde, UPS substratın kararlılık, hücre içermeyen bir sistem içinde, böyle bir F-box proteini olarak proteozomal bozunum yolu, temel bileşenlerinden biri varlığında veya yokluğunda analiz edilir. Genel olarak, bu dokulardan hücresiz özleri hazırlamak ve western blotting ile ilgilenilen protein (ler) in miktarları, monitör bitki dokularında test edilen protein (ler) ifade eder. Protein degradasyon UPS-bağımlı mekanizma özel proteozomal inhibitörleri ve / veya bir bileşen SCF, cullin dominant-negatif bir form of yakımının birlikte kullanılarak dahil ile gösterilmiştir. Biz VBF 15 F-box proteini ile, Arabidopsis VIP1 17 protein proteozomal bozundurması kullanılarak, bu tahlili göz önüne alarak, bu da başka alt-tabakalar proteozomal stabilitesini incelemek için kullanılabilir.

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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Protein assay kit</td>
			<td style="width:129px;">Bio-Rad</td>
			<td style="width:119px;">500-0001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Proteinase inhibitor cocktail&#160;</td>
			<td style="width:129px;">Sigma-Aldrich</td>
			<td style="width:119px;">S8820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Mini-Protean system</td>
			<td style="width:129px;">Bio-Rad</td>
			<td style="width:119px;">165-8000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Semi-dry western blotting SD electrotransfer system</td>
			<td style="width:129px;">Bio-Rad</td>
			<td style="width:119px;">170-3940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Affinity Purified Rabbit Anti-Ha</td>
			<td style="width:129px;">icllab</td>
			<td>RHGT-45A-Z</td>
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			<td height="42" style="height:42px;width:247px;">Goat anti-Rabbit IgG Peroxidase Conjugate</td>
			<td style="width:129px;">Thermo Scientific</td>
			<td style="width:119px;">31460</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:247px;">BioTrace, NT nitrocellulose transfer membrane</td>
			<td style="width:129px;">Pall Corportation</td>
			<td style="width:119px;">27377-000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Immobilon western chemiluminescent HRP substrate</td>
			<td style="width:129px;">EMD Millipore</td>
			<td style="width:119px;">WBKL S0 050</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">MG132</td>
			<td style="width:129px;">EMD Millipore</td>
			<td>474790-1MG</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Lactacystin</td>
			<td style="width:129px;">Sigma-Aldrich</td>
			<td>L6785</td>
			<td></td>
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			<td height="42" style="height:42px;width:247px;">Thermo Scientific Pierce Fast Western Blot Kit, ECL Substrate</td>
			<td style="width:129px;">Pierce</td>
			<td style="width:119px;">35055</td>
			<td></td>
		</tr>
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assaying proteasomal degradation in a cell-free system in plants

Protein bozulması için ubikuitin-proteazom yolunun hemen hemen tüm ökaryotik organizmalarda hücresel fonksiyonların geniş bir yelpazede düzenlenmesi için en önemli mekanizmalardan biri olarak ortaya çıkmıştır. Özellikle bitkilerde, ubiquitin/26S proteazom sistemi (UPS) protein bozulmasını düzenleyen ve bağışıklık tepkisi, geliştirme ve programlanmış hücre ölümü gibi işlemler geniş bir yelpazede, geliştirilmesine önemli ölçüde katkıda bulunur. Ayrıca, artan kanıtlar Agrobacterium gibi çok sayıda bitki patojenleri, bitki-patojen etkileşimleri UPS önemini vurgulayarak, etkin enfeksiyon konak UPS istismar olduğunu göstermektedir. UPS alt-tabaka özgüllüğü, E1 ile uyum içinde hareket eder ve E2 bunlara ubiquitin moleküllerin zincirleri takılarak bozulması için hedeflenen spesifik protein moleküllerini tanıyan ve işaretlemek için ligaz E3 ligaz ubikitin ile elde edilir. E3 ligazlar bir sınıfı olan SCF (SKP1 / CÖzellikle UPS substratları tanır ve kendi F-box protein bileşeni aracılığıyla ubikuitinasyomınun için onları hedef Ullin / F-box protein) kompleksi. Ilgi konusu bir biyolojik süreç içinde UPS bir potansiyel rolünü araştırmak için, UPS aracılığında protein indirgenmesi için basit ve güvenilir bir tahlil hazırlamak için önemlidir. Burada, bir bitki hücresi serbest sistemi kullanılarak böyle bir tahlili tarif eder. Bu deney, F-box protein-substrat etkileşimleri ile ilgili özel bir odaklanarak, çeşitli hücresel süreçlerde düzenlenmiş protein degradasyon rolleri çalışmalar için uyarlanabilir.

Zaltsman et al. 17 uyarlanmıştır Şekil 1, bir hücre barındırmayan sistemde proteozomal bozulmasının saptanması için örnek deneyleri gösterir. Özellikle, N. SCF VBF yolu aracılığıyla VBF F-box proteini tarafından bir bitki savunma-ilişkili protein VIP1 istikrarsızlaştırma göstermek benthamina. Arabidopsis VBF ve HA-etiketli VIP1 (HA-VIP1) geçici proteinler eksprese edildi ve ifade yaprak ekstreleri içinde HA-VIP1 içerik ...

Watch this Scientific Journal Video about Assaying Proteasomal Degradation in a Cell-free System in Plants at JoVE.com

Assaying Proteasomal Degradation in a Cell-free System in Plants

Hedeflenen protein yıkımı, hücre fonksiyonu için önemli bir düzenleyici mekanizmasını temsil eder. Daha sonra, 26S proteazomu için moleküler &quot;etiketleri&quot; hizmet hedef proteine ​​bağlanan bir Poliübikütin zincirleri korunmuş ubikitin-proteazom süreci yoluyla gerçekleşir. Burada, proteinlerin proteozomal indirgenmesi için basit ve güvenilir bir hücre içermeyen tahlili tarif eder.

Bitkilerde bir Cell-serbest Sistemde proteozomal indirgenme Assaying

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

assaying-proteasomal-degradation-in-a-cell-free-system-in-plants

Research

JoVE Journal

Biology

14.3K Görüntüleme.  Stony Brook University, State University of New York.  Hedeflenen protein yıkımı, hücre fonksiyonu için önemli bir düzenleyici mekanizmasını temsil eder. Daha sonra, 26S proteazomu için moleküler "etiketleri" hizmet hedef proteine ​​bağlanan bir Poliübikütin zincirleri korunmuş ubikitin-proteazom süreci yoluyla gerçekleşir. Burada, proteinlerin proteozomal indirgenmesi için basit ve güvenilir bir hücre içermeyen tahlili tarif eder. 

Video: Assaying Proteasomal Degradation in a Cell-free System in Plants

Maintaining Wolbachia in Cell-free Medium

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ascertain the viability of the purified Wolbachia and (3) maintain the now extracellular Wolbachia in cell-free medium.  Purified Wolbachia remain alive in the extracellular phase but do not replicate until re-inoculated into eukaryotic cells.  Extracellular Wolbachia purified in this manner will remain viable for at least a week at room temperature, and possibly longer.  Purified Wolbachia are suitable for micro-injection, DNA extraction and other applications. 


This video describes a method for purifying Wolbachia pipientis from an Anopheles gambiae cell line and then culturing the endosymbiont in cell-free medium. An assay for viability of the bacterium is demonstrated.

Hücre Orta Wolbachia bakımı

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ...

Biyoloji

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing: Doğru Genotiplendirme için Basit Bir Yöntem

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Küçük Molekül İndüksiyon Pluripotent İnsan Embriyonik Kök Hücreleri İnsan Kardiyak Öncüleri ve kardiyomiyositlerde Verimli türetilmesi

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

Içinde Postembryonic Fenotiplerin Belirleme RNAi Tarama C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

LRRK2 Kinaz Aktivitesi Assaying In vitro</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

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 ...

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

Bir uygulama için Protokoller<em&gt; Escherichia coli</emSentetik Biyoloji&gt; Tabanlı TX-TL Hücre-Özgür İfade Sistemi

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, &beta;-catenin. Two different methods are described to assess &beta;-catenin protein degradation in Xenopus egg extract. One method is visually informative ([35S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess &beta;-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of &beta;-catenin degradation.

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

Β-katenin Bozulması Of Sulandırma<em&gt; Xenopus</em&gt; Yumurta Özü

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Tek kanallı bir hücre-bağlı Patch-clamp kayıt

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Bitkilerde bir Cell-serbest Sistemde proteozomal indirgenme Assaying

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Β-katenin Bozulması Of Sulandırma

Tek kanallı bir hücre-bağlı Patch-clamp kayıt