Biz bize florasan spektrometre kullanmak için izin verdiği için ve onun yardım için Dr Thierry Pourcher minnettarız. Laboratuvar Ligue Nationale Contre le Cancer (équipe labellisée) tarafından finanse edilen, Dernek la Recherche contre le Kanser, Institut National du Yengeç (Inca) dökmek, Agence Nationale la Recherche, METOXIA (AB programı FP7), Centre dökmek A. Lacassagne, Centre National de la Recherche Scientifique, Institut National de la Sante et de la Recherche Médicale ve Nice Üniversitesi ( <a href="http://www.unice.fr/isdbc/" target="_blank">http://www.unice.fr/isdbc/</a> ). Biz eleştirel okuma ve editoryal düzeltme için Dr M Christiane Brahimi-Horn teşekkür ederim.

Glikojenin Biyokimyasal titrasyonu 1.. Cell Lysis <ol><li> 100 mm çaplı tabak başına 0.5-2 x 10 6 konsantrasyonunda tohum hücreleri. </li><li> Tedavi: hücreleri inkübe 24,% 1 veya% 0.1 O 2,% 94 ya da ayarlanmış bir Bug-Box anaerobik çalışma istasyonu (Ruskinn Teknoloji Biotrace International Plc, Bridgend, UK) düşük oksijen konsantrasyonu (hipoksi) 48 ya da 72 saat % 94.9 N 2 ve% 5 CO 2. Buna paralel olarak, (% 21 O 2,% 5 CO2) normoksiyada hücreleri inkübe edin. Deney zamanında glikoz konsantrasyonundaki değişimleri en aza indirmek için, her 24 saat (25 mM glikoz-içeren ortam) ortam değiştirin. </li><li> Tedaviden sonra hücreler, yıkama kültür ortamından glukoz izlerini çıkarmak için PBS ile 2 kat. Soğuk PBS hücreleri kazıyın. </li><li> 5 dakika boyunca 1000 rpm&#39;de santrifüje çözeltisi, süpernatantı atmak ve bir kez PBS ile pelet yıkayın. Pelet fazla ilerlemeden önce -20 ° C&#39;de dondurulmuş olabilir.</li><li> Damıtılmış su, 100 ul pelet tekrar. Glikojen Assay Kit (Abcam) ile sağlanan glikojen Hidroliz Tampon 100 ul ekleyin. Enzimleri inaktive etmek için 5 dakika boyunca 95 ° C &#39;de lizat kaynatın. </li><li> Çözünmeyen ürünleri çıkarmak için 10 dakika boyunca 13,000 rpm&#39;de Vorteks ve santrifüj lizat. Süpernatant tutun. </li><li> Örnekler arasındaki protein içeriğine glikojen seviyeleri normalleştirmek için, proteinlerin ölçümü bisinkoninik asit yöntemi (BCA-Interchim) kullanılarak 25-35 ul süpernatan ile yapılabilir. </li></ol> Lizat bazı hücrelerin içinde serbest glükoz konsantrasyonunu ölçmek için kullanılabilir. 2. Glikojen hidrolizi <ol><li> Hidroliz Enzim Karışımı 1 ul süpernatan (aşama 1) 50 ul karıştırın. Bu karışım glukoamilazı içerir. Oda sıcaklığında 30 dakika boyunca inkübe edin. </li><li> Standart glikojen seyrelterek, kalibrasyon eğrisinin hazırlanması (2 mg / ml) shidroliz tamponu içinde 10 ug / ml &#39;lik bir solüsyonuna kiti ile upplied. Daha sonra, altı ayrı 0 ile tüpler, 4, 8, 12, 16, ve 10 ug / ml bir standart 20 ul hazırlamak ve 0 glikojen bir konsantrasyon vermek üzere hidroliz tampon maddesi ile 50 ul&#39;lik nihai bir hacme kadar her bir tüp ayarlamak 0.04 , 0.08, 0.12, 0.16 ve 0.2 ug / ml. Standartlarda hidrolizi Enzim Karışımı 1 ul ilave edin ve oda sıcaklığında 30 dakika boyunca inkübe edin. </li></ol> Serbest glukoz konsantrasyonu için değer ile verilen hücre çizgisinin arka glikoz konsantrasyonu, hücre glikojen seviyesini belirlemek için toplam glikoz konsantrasyonu (glikojen hidroliz + serbest-glikozdan, glikoz) için değerden çıkartılır gerekir. 3. Kalkınma ve Floresans Okuma <ol><li> Her bir durumda, serbest glükoz konsantrasyonu ve iki boruyu ölçmek için (boru 1) bir tüp hazırlanması (boru 2 ve 3), toplam glikoz konsantrasyonunu (her boru wit ölçmekhidrolize glikojen ha farklı ses). </li><li> Borunun 1 içinde 1 aşamasının sonunda ekstre süpernatan 15 ul ekle. 50 ul bir son hacme tamamlamak için hidroliz tamponu 35 ul ekle. Elde edilen floresan, serbest glükoz konsantrasyonunu verir. </li><li> Tüp 2 (adım 2&#39;de elde edilmiştir) hidroliz glikojen X ul ekleyin. 50 ul bir son hacme kadar ayarlayın. Numune hacmi (X ul) kalibrasyon eğrisinin konsantrasyonları dışında değildir floresans değerler elde etmek amacıyla hücre yoğunluğu ve / veya hücre tipine göre ayarlanmalıdır. </li><li> Borunun 3 içinde hidrolize glikojen 2 X ul ekleyin. 50 ul bir son hacme kadar ayarlayın. </li><li> Her tüp için Kalkınma Tampon 48.7 ul, Kalkınma Enzim Mix 1 ul ve (Glikojen Assay Kit verilen) OxiRed Probe 0.3 ul karıştırılarak numuneler ve standartlar için bir geliştirme çözüm hazırlayın. Örneğin, 2 koşullar için, 12 borusu için bir karışımını hazırlamaks (standartları 6, serbest glükoz 2 ve toplam glikoz floresan 4). </li><li> Her tüpe bu karışımı 50 ul eklenir ve oda sıcaklığında karanlıkta 30 dakika boyunca inkübe edin. </li><li> Tavsiye edildiği gibi 535 nm, 587 nm&#39;lik bir emisyon dalga boyunda bir uyarma dalga boyunda floresan ve bir eksitasyon ve emisyon için 3 nm&#39;lik bir yarık ölçün. </li></ol>

<ol>
	<li>Pelletier, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+Synthesis+is+Induced+in+Hypoxia+by+the+Hypoxia-Inducible+Factor+and+Promotes+Cancer+Cell+Survival.">Glycogen Synthesis is Induced in Hypoxia by the Hypoxia-Inducible Factor and Promotes Cancer Cell Survival.</a> Front Oncol. 2, (2012).</li><li>Alonso, M. D., Lomako, J., Lomako, W. M., Whelan, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+look+at+the+biogenesis+of+glycogen.">A new look at the biogenesis of glycogen.</a> FASEB J. 9, 1126-1137 (1995).</li><li>Bollen, M., Keppens, S., Stalmans, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+features+of+glycogen+metabolism+in+the+liver.">Specific features of glycogen metabolism in the liver.</a> Biochem. J. 336 (Pt 1), 19-31 (1998).</li><li>Parker, G. J., Koay, A., Gilbert-Wilson, R., Waddington, L. J., Stapleton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMP-activated+protein+kinase+does+not+associate+with+glycogen+alpha-particles+from+rat+liver.">AMP-activated protein kinase does not associate with glycogen alpha-particles from rat liver.</a> Biochem. Biophys. Res. Commun. 362, 811-815 (2007).</li><li>Sullivan, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature+of+alpha+and+beta+particles+in+glycogen+using+molecular+size+distributions.">Nature of alpha and beta particles in glycogen using molecular size distributions.</a> Biomacromolecules. 11, 1094-1100 (2010).</li><li>Chee, N. P., Geddes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+liver+glycogen.">The structure of liver glycogen.</a> FEBS Lett. 73, 164-166 (1977).</li><li>Roach, P. J., Depaoli-Roach, A. A., Hurley, T. D., Tagliabracci, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+and+its+metabolism:+some+new+developments+and+old+themes.">Glycogen and its metabolism: some new developments and old themes.</a> Biochem. J. 441, 763-787 (2012).</li><li>Moses, S. W., Bashan, N., Gutman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+metabolism+in+the+normal+red+blood+cell.">Glycogen metabolism in the normal red blood cell.</a> Blood. 40, 836-843 (1972).</li><li>Agbanyo, M., Taylor, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+3-deoxy-3-fluoro-D-glucose+into+glycogen+and+trehalose+in+fat+body+and+flight+muscle+in+Locusta+migratoria.">Incorporation of 3-deoxy-3-fluoro-D-glucose into glycogen and trehalose in fat body and flight muscle in Locusta migratoria.</a> Biosci. Rep. 6, 309-316 (1986).</li><li>Louzao, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Fluorescent+glycogen"+formation+with+sensibility+for+in+vivo+and+in+vitro+detection.">"Fluorescent glycogen" formation with sensibility for in vivo and in vitro detection.</a> Glycoconj. J. 25, 503-510 (2008).</li><li>Sheehan, D. C. H., B, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Theory and practice of histotechnology. , (1980).</li><li>Baba, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+monoclonal+antibody+that+recognizes+glycogen+and+its+application+for+immunohistochemistry.">Production of monoclonal antibody that recognizes glycogen and its application for immunohistochemistry.</a> Kokubyo Gakkai Zasshi. 60, 264-287 (1993).</li></ol>

Yazarlar herhangi bir çıkar çatışması beyan ederim.

In vitro olarak glikojen biyokimyasal titrasyon hücreleri glikojen içerik doğru bir ölçümü sağlar. Diğer bazı teknikler (bir glikojen antikor ile PAS, immunofloresans, vb.) Ile karşılaştırıldığında, bu titrasyon çok özel, hassas ve tekrarlanabilir. Bu radyoaktivite, ancak florasan spektrometre gerektirdiğinden Ayrıca, yöntem uygundur. Ancak, bu teknik, nicel ve hücre içinde glikojen dağılımı hakkında bilgi sağlamaz. Bu yazıda tarif edilen tek...

Glikojen pek çok hücre sitoplazması içinde mevcut olan glikoz kalıntısının bir multibranched polimerdir. Bu hücrelerde enerji depolama ana biçimlerinden biridir ve glikoz metabolizmasında önemli bir rol oynar. Çoğu memeli hücreleri hızla metabolik stres sırasında glikoliz ve ATP üretimini teşvik etmek için glikoz içine bozulmuş olabilir ve mağaza glikojen, üretebiliriz. Hepatositler böylece vücuda glikoz sürekli tedarik sağlayan kan şekeri seviyesini düzenlemek için glikojen büyük miktarda üretir. Bunun aksine, diğer hücreler (kas, kırmızı kan hücreleri, vs) glikojen konsantrasyonu, nispeten düşüktür. Bununla birlikte, lokal olarak, bu miktarlar hücreler birden besin yoksun bir ortama maruz kaldıklarında kısa vadede enerji sağlamak için yeterlidir. Glikojen sentezi ve glikojen dökümü tüm dokularda aynı adımlar (Şekil 1) takip eder. İlk olarak, glikozglikoz taşıyıcılannın (gluts) ile hücrelere girer ve hızla fosfoglukomutaz glikoz-1-fosfat (G1P) glukoz-6-fosfat (G6P) dönüştürülür. G1P sonra UDP-glikoza modifiye edilir ve UDP-glikoz karbon C1 glycogenin, glikojen ankraj protein tirosin artığı bağlanmıştır. Bu molekül, bir glikojen primer olarak, glikojen sentaz üzerinden bir α (1 → 4) bağı ile terminal glikoz için UDP-glikoz eki ile uzatılır. 11 den fazla glikoz kalıntısının doğrusal bir zincir meydana getirildiği zaman, son olarak, dallanma enzimi, bir at α (1 → 6) bağı vasıtasıyla bir zincirin glikoz 6 glikoz kalıntısı, en az oluşan bir terminal oligosakkarit aktarır. Bu işlemin tekrarlanması tur başına 6.5 glükoz ile bir sarmal oluşturmak dalları ihtiva eden büyük bir fraktal bir yapı verir. Glikojen uyumlu eylem glukoza ters hidrolize olabilirBir dalın glikoz son artık ile glikojen molekülü arasındaki sınır glikosidik α (1 → 4) hidrolize α (1 → 6) bağlı ve glikojen fosforilaz hidrolize eden enzimler dallara ayrılmayı giderme bölgesinin. Glikojenolizin olarak bilinen bu reaksiyon, (ATP tüketimine yansıtan) AMP seviyelerinin arttırılması ile aktive ve glikoz ve ATP 2,3 ile inhibe edilir. Elektron mikroskobu ile, glikojen molekülü bir çok hücre tipinde çapı 15-30 nm olarak β serbest parçacıklar (ya da glikojen monopartıcles) &#39;de tarif edilmiştir. Hepatositler gibi özel hücre tiplerinde, β parçacıklar 80 nm ila 200 nm, 4, maksimum çapı da değişebilir α partiküller olarak bilinen rozetler, oluşturulması için bir kompleksi içine monte edilebilir </sup&gt;. Bu parçacıklar β α parçacıkların büyük kümeler oluşturmak için bağlı oldukları şekilde hala tam olarak aydınlatılamamıştır. Bazı kanıtlar β parçacıklar kovalent bağlama 5, hidrojen bağlanması, ya da protein-protein etkileşimleri yoluyla 6 ile bağlanmış olabilir kanıtlamak eğilimindedir. Hücrelerinde depolanmış olan glikojen miktarı, bir çok parametreye bağlıdır: (I) glikojen sentezini başlatır hücrede glycogenin miktarı, protein fosforilasyon / defosforilasyon düzenlenir glikojen sentaz ve fosforilazın (II) aktivitesi, (III) konsantrasyonu örneğin damar sistemi glukoz kaynağı ve hücreleri tarafından glikoz alımının gibi çeşitli parametrelere bağlıdır hücrelerin, glikoz. Glikojen depoları sıkı enerji metabolizmasını düzenleyen hormonlar tarafından, ara metabolitleri yoluyla biyosentetik hormonların allosterik yönetmelikle düzenlenir ve besin algılama sinyal yollarının 7 tarafından edilir. Bu olmak önemlidirdaha tüm vücut ve hücresel düzeyde glikojen metabolizmasının önemini anlamak için biyolojik numunelerde glikojen ölçmek mümkün. Biz burada glikojen için in vitro deneyinde bir hassas, yeniden üretilebilir ve uygun biyokimyasal açıklar. Bu teknik, glikojen özel hidroliz öncesi ve sonrası glikoz miktar floresan dayanmaktadır. Diğer yöntemler hücrelerde glikojen seviyesini tahmin etmek vardır, ama bunların çoğu kantitatif değildir. Hücrelerde glikojen ölçümü için tarif edilen birinci tekniklerden biri glikojen 8,9 halinde [14C]-glükoz dahil ölçümüne dayanıyordu. Radyoaktivite kullanılması işlemek için bu işlem daha zor hale getirir ama glikojen içine ve dış dallarına ve molekülün çekirdek glikoz kalıntısının dağılımı arasında ayırt glikoz dahil oranını temin etme avantajına sahiptir (aynı zamanda bir gerektirir Ek β-amylolysis ve kromatografik adım). Başka bir teknik, daha yakın zamanda geliştirilmiştir ve 2-NBDG eklenmesi dayanır (2 - {N-[7-nitrobenz-2-oksa-1 ,3-diazol-4-il] amino}-2-deoksiglukoz), bir glikojen 10 içine 2-deoksiglukoz floresan türevi. Ölçülen floresans yoğunluğu üretilen glikojen miktarını yansıtır ve bir floresan okuyucu ile ölçülebilir. Hücredeki floresan dağılımı da konfokal mikroskopi ile değerlendirilebilir. Diğer nonquantitative teknikler arasında, Periyodik asit-Schiff boyama (PAS) belki de en yaygın olanıdır. Bu, sabit hücrelerde, parafin içinde doku bölümleri ya da dondurulmuş olarak glikojen tespiti için de kullanılabilir. Mor Bu histolojik teknik renkler olmayan özel polisakkaritler, glikolipitler, glikoproteinler, selüloz ve nötr müsinler. Bu testin özgüllüğü özel olarak glikojen digests diastaz, sabit hücre veya doku kesitlerinin tedavisi ile arttırılabilir. Daha sonra,glikojen nitel düzeyi (karbonhidrat dışında glikojen makro molekülleri modifiye edilmiş) hidrolize örnekler hidrolize olmamış örnekleri (her karbonhidrat değiştirilmiş makro moleküller) karşılaştırılması ile tahmin edilebilir. Glikojen biyokimyasal deneylerde farklı PAS boyanması ve mikroskopik analizleri, dağınık ya da hücrenin belirli bir kısmı içinde konsantre edilebilir hücrede glikojen dağılımı hakkında bilgi sağlar. Hatta farklı koşullar arasında glikojen birikiminin PAS boyama tahminler farklar da, ancak, bu 11 kantitatif değildir. Bir monoklonal fare antikoru başlangıçta in vitro antijen 12 hücrelerinde glikojen ve saflaştırılmış glikojen ile tepkimeye gösterildiği gibi çene kondiler kıkırdak kullanılarak yapılır. Bu antikor özel olarak glikojen ilgili şeker zincirleri kabul ettiği gibi, bu PAS boyanması daha özel bir şekilde immünohistokimya ile glikojen tespiti için yararlı bir araçtır. </p&gt; Elektron mikroskopisi hücreleri ve glikojen depolama derecesinin değerlendirilmesinde glikojen tanelerinin görüntülenmesini sağlayan bir başka tekniktir. Aslında, glikojen β parçacıkların elektron yoğun granüller 1 gibi bir elektron mikroskobu ile kolayca tanınabilir.

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			<td></td>
			<td></td>
			<td>REAGENTS</td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>31966.047</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Glycogen Assay Kit</td>
			<td>Abcam</td>
			<td>ab65620 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		<tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
	 </tr>
		 <tr>
			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments (optional)</td>
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		 <tr>
			<td>Fluorescence Spectrometer</td>
			<td>PerkinElmer</td>
			<td>LS 50B</td>
			<td></td>
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biochemical titration of glycogen in vitro

Glikojen omurgalı hayvanlarda glikoz ana enerjik polimerdir ve önemli bir tüm vücut metabolizmasında rol hem de hücresel metabolizmada oynar. Glikojen tespit etmek için birçok yöntem zaten var ama sadece birkaç nicel vardır. Burada glikoamilaz glukoza glikojen özel bozulması dayanır Abcam Glikojen deney takımı kullanılarak bir metodu tarif etmektedir. Glukoz sonra özellikle floresan üretmek için OxiRed prob ile reaksiyona giren bir ürün için oksitlenir. Titrasyon doğru, hassas ve hücre ekstreleri veya doku bölümleri üzerinde elde edilebilir. Bununla birlikte, diğer tekniklerin aksine, bu hücrede glikojen dağılımı hakkında bilgi vermemektedir. Bu tekniğin bir örneği olarak, normoksiyada kuluçkaya iki hücre hatları, Çin hamsteri akciğer fibroblast CCL39 ve insan kolon karsinoma LS174, hipoksi karşı (% 21 O 2) (1% 2 O) burada glikojen titrasyonu açıklar. Biz hipoksi bir sig olduğunu varsaydıknal o hücreleri 1, hayatta kalmak için sentez ve mağaza glikojen hazırlar.

Tümör hücrelerine karşı tümörler sinyaller oksijen (hipoksi) hayatta kalmak üzere, daha sonra besin tüketimi işlemek için enerji depolamak için ihtiyaç bir düşük düzeyde. Glikojen memeli hücrelerinde glikoz ana enerji polimer olduğu için, hipoksi glikojen depolama düzenleme incelenmiştir. Tablolar 1, 2 ve 3 &#39;de gösterildiği gibi, hücre lizatları içinde glikojen konsantrasyonunun hesaplanması ve standardizasyon floresans ham veriler üzerinde gerçekleşti...

Watch this Scientific Journal Video about Biochemical Titration of Glycogen In vitro at JoVE.com

Biochemical Titration of Glycogen In vitro

Burada in vitro glikojen titrasyonu için bir doğru, tekrarlanabilir ve uygun biyokimyasal yöntem açıklanmaktadır. Bu teknik Abcam Glikojen deney kiti kullanır ve floresan ile titrasyon glikoz ve glikoz glikojen ardışık hidrolizi dayanmaktadır.

Glikojenin Biyokimyasal titrasyonu<em&gt; In vitro</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

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Biology

Biochemical Titration of Glycogen In vitro

28.3K Görüntüleme.  University of Nice - Sophia Antipolis.  Burada in vitro glikojen titrasyonu için bir doğru, tekrarlanabilir ve uygun biyokimyasal yöntem açıklanmaktadır. Bu teknik Abcam Glikojen deney kiti kullanır ve floresan ile titrasyon glikoz ve glikoz glikojen ardışık hidrolizi dayanmaktadır. 

Video: Biochemical Titration of Glycogen In vitro

Titration of Human Coronaviruses Using an Immunoperoxidase Assay

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for prototype strains 229E and OC43 of human coronavirus (HCoV). Therefore, an alternative indirect immunoperoxidase assay (IPA) was developed for the detection and titration of these viruses and is described herein. Susceptible cells are inoculated with serial logarithmic dilutions of virus-containing samples in a 96-well plate format. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as 'Tissue Culture Infectious Dose 50 percent' (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain infectious replicating virus. This technique provides a reliable method for the titration of HCoV-229E and HCoV-OC43 in biological samples such as cells, tissues and fluids. This article is based on work first reported in Methods in Molecular Biology (2008) volume 454, pages 93-102.

In this video, we demonstrate an alternative method for detection and titering of viruses using an enzymatic antigen detection technique known as an immunoperoxidase assay. Here, we will show you how to collect your viral samples, prepare the cells for testing, and finally the immunoperoxidase assay using serial dilutions to determine the viral titer.

Immunoperoksidaz Testi İnsan coronavirüsleri Titrasyon

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for ...

Biyoloji

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Zebra balığı Beyin Ventrikül Enjeksiyon

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host macromolecule. ITC has some advantages over common spectroscopic approaches for studying host/ligand interactions. For example, the heat released or absorbed when the two components interact is directly measured and does not require any exogenous reporters. Thus the binding enthalpy and the association constant (Ka) are directly obtained from ITC data, and can be used to compute the entropic contribution. Moreover, the shape of the isotherm is dependent on the c-value and the mechanistic model involved. The c-value is defined as c = n[P]tKa, where [P]t is the protein concentration, and n is the number of ligand binding sites within the host. In many cases, multiple binding sites for a given ligand are non-equivalent and ITC allows the characterization of the thermodynamic binding parameters for each individual binding site. This however requires that the correct binding model be used. This choice can be problematic if different models can fit the same experimental data. We have previously shown that this problem can be circumvented by performing experiments at several c-values. The multiple isotherms obtained at different c-values are fit simultaneously to separate models. The correct model is next identified based on the goodness of fit across the entire variable-c dataset. This process is applied here to the aminoglycoside resistance-causing enzyme aminoglycoside N-6'-acetyltransferase-Ii (AAC(6')-Ii). Although our methodology is applicable to any system, the necessity of this strategy is better demonstrated with a macromolecule-ligand system showing allostery or cooperativity, and when different binding models provide essentially identical fits to the same data. To our knowledge, there are no such systems commercially available. AAC(6')-Ii, is a homo-dimer containing two active sites, showing cooperativity between the two subunits. However ITC data obtained at a single c-value can be fit equally well to at least two different models a two-sets-of-sites independent model and a two-site sequential (cooperative) model. Through varying the c-value as explained above, it was established that the correct binding model for AAC(6')-Ii is a two-site sequential binding model. Herein, we describe the steps that must be taken when performing ITC experiments in order to obtain datasets suitable for variable-c analyses.

ITC is a powerful tool for studying the binding of a ligand to its host. In complex systems however, several models may fit the data equally well. The method described here provides a means to elucidate the appropriate binding model for complex systems and extract the corresponding thermodynamic parameters.

Cilt Mekanizmaları belirleme Sipariş Değişken konsantrasyon İzotermal Titrasyon Kalorimetre Veri Kümeleri toplanıyor

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

Yetişkin Zebra balığı Biyokimyasal Analiz için Kan Alma

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

Fat Kütle Biyokimyasal ve Yüksek Verimli Mikroskobik Değerlendirme<em&gt; Caenorhabditis Elegans</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

ATP-bağımlı Kromatin Tadilat Enzimler Faaliyetlerinin Analizi için biyokimyasal Tahliller

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

Lusiferaz ifade eden rekombinant virüsler yüksek verimlilik Titrasyon

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

Büyüme-tabanlı Belirlenmesi ve Protein Parçalanabilirliklerinin Genetik Talepler biyokimyasal Onay<em&gt; Saccharomyces cerevisiae</em

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

Genetik ve biyokimyasal Yaklaşımlar<em&gt; İn Vivo</em&gt; ve<em&gt; İn Vitro</em&gt; Protein oligomerization Değerlendirilmesi: Riyanodin Reseptör Vaka Çalışması

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

Saccharomyces cerevisiae Exponential Growth Kinetics in Batch Culture to Analyze Respiratory and Fermentative Metabolism

Saccharomyces cerevisiae cells in the exponential phase sustain their growth by producing ATP through fermentation and/or mitochondrial respiration. The fermentable carbon concentration mainly governs how the yeast cells generate ATP; thus, the variation in fermentable carbohydrate levels drives the energetic metabolism of S. cerevisiae. This paper describes a high-throughput method based on exponential yeast growth to estimate the effects of concentration changes and nature of the carbon source on respiratory and fermentative metabolism. The growth of S. cerevisiae is measured in a microplate or shaken conical flask by determining the optical density (OD) at 600 nm. Then, a growth curve is built by plotting OD versus time, which allows identification and selection of the exponential phase, and is fitted with the exponential growth equation to obtain kinetic parameters. Low specific growth rates with higher doubling times generally represent a respiratory growth. Conversely, higher specific growth rates with lower doubling times indicate fermentative growth. Threshold values of doubling time and specific growth rate are estimated using well-known respiratory or fermentative conditions, such as non-fermentable carbon sources or higher concentrations of fermentable sugars. This is obtained for each specific strain. Finally, the calculated kinetic parameters are compared with the threshold values to establish whether the yeast shows fermentative and/or respiratory growth. The advantage of this method is its relative simplicity for understanding the effects of a substance/compound on fermentative or respiratory metabolism. It is important to highlight that growth is an intricate and complex biological process; therefore, preliminary data from this method must be corroborated by the quantification of oxygen consumption and accumulation of fermentation byproducts. Thereby, this technique can be used as a preliminary screening of compounds/substances that may disturb or enhance fermentative or respiratory metabolism.

Saccharomyces cerevisiae Exponential Growth Kinetics in Batch Culture to Analyze Respiratory and Fermentative Metabolism

Here we present a protocol to estimate the respiratory and fermentative metabolism by fitting the exponential growth of Saccharomyces cerevisiae to the exponential growth equation. Calculation of the kinetic parameters allows for the screening of influences of substances/compounds on fermentation or mitochondrial respiration.

Saccharomyces cerevisiae Üstel büyüme kinetik solunum ve fermantatif metabolizma analiz etmek için toplu iş kültüründe

Saccharomyces cerevisiae cells in the exponential phase sustain their growth by producing ATP through fermentation and/or mitochondrial respiration. The ...