Bu çalışma, Kanada Sağlık Araştırma Enstitüsü (CIHR), Ulusal Bilim ve Mühendislik Araştırma Konseyi (NSERC) ve bir CIHR eğitim bursu bursu (LF) tarafından desteklenmiştir. Biz AAC için Prof. Gerard D. Wright (McMaster Üniversitesi, Kanada) (6)-Ii ifade plazmid teşekkür ederiz.

1. Stok çözelti hazırlanması <ol><li> Ilgi makromolekül arındırın. (Bu durumda, başka rapor gibi aminoglikozid N-6&#39;-asetiltransferaz-Ii (AAC6&#39; Ii), izole edilmiştir. 13) </li><li> 4 litre diyaliz tampon hazırlayın. (Bu durumda 25 mM 4 - (2-hidroksi)-1-piperazineethanesulfonic asit (Hepes MW 238,3 g / mol), 2 mM ethylenediaminetetraacetic asit (EDTA, 292,2 MW) içeren, pH 7.5.) </li><li> Protein AAC (6 &#39;)-Ii örneği (400 mcM az 5 ml) kullanılan diyaliz tampon (3 x 1.3 L) diyalize olmalıdır Dialyze. Son diyaliz solüsyonu makine durulama ve örnekler sulandırmak için tutulur. </li><li> 0.45 mikron selüloz filtre ile son diyaliz solüsyonu Filtresi ve 4 ° C&#39;de saklanan &#39;Çalışan tampon&#39; olarak sevk edilecektir </li><li> Akan tamponu ile iyice durulanması, 0.2 mikron şırınga filtre süzülür protein çözüm. Standart bir test (Bradford, Lowry, vb) veya UV absorbans kullanarak protein nihai konsantrasyonu ölçün. Uzun vadeli istikrar en üst düzeye çıkarmak için protein saklayın. (AAC (6 durumda)-Ii dondurma ve çözme sonra faaliyet korumak değildir &#39;)-Ii, bu depolama 4 ° C AAC (6 anlamına gelir&#39;). </li><li> Tampon çalışan 4,0 mg eriterek asetil koenzim A 25 mM stok solüsyonu (AcCoA, ligand, 809,57 MW) 200μL hazırlayın. , -78 Anda Freeze ° C Kullanıma hazır olana kadar. </li><li> Tüm protein ve ligand örnekleri, konsantrasyon rastgele örnek-örnek dalgalanmaları en aza indirmek için, aynı stok çözümleri geliyor olmalıdır. Tüm değişken-c veri kümesi üzerinde protein konsantrasyonu için tek bir düzeltme faktörü analiz ayarlanır. 10 </li></ol> 2. ITC numunelerin hazırlanması <ol><li> 2 mL nihai hacmi ile 64 arasında bir c değeri enzim çözeltisi seyreltilir. (AAC (6 &#39;)-Ii olması durumunda, bu 192 mcM karşılık gelir.) </li><li> Buz su ligand (AcCoA) stok solüsyonu hızla çözünme. </li><li> Bir AcCoA çözüm 0.5 ml 10 kat daha fazla konsantre sonra bu durumda protein bağlayıcı sitelerinin sayısı, tampon çalışan 420 mcL AcCoA stok solüsyonu 80 mcL seyreltilmesi ile 4 mM. Hazırlayın Bir pipetle dolum tüpü içine aktarın. Hızla -78 ° C AcCoA stok solüsyonu geri </li><li> Degas bir sıcaklık 1 ile 5 dakika boyunca protein ve vakum altında çözümler ° C istenilen çalışan sıcaklık altında (19 ° C). </li></ol> 3. Şırınga ayarlama 14 <ol><li> Önceden monte edilmiş şırınga kelepçe sahibi olarak aynı yüksekliğe kadar şırınga tutucu ile enjeksiyon şırınga takın. </li><li> Şırınga sahibinin alt sıkıca bastırılır kadar enjeksiyon şırınga üzerinden ikinci şırınga kelepçe besleyin. Sağlanan 0.050 &quot;Tükenmez Hex Sürücü ile kelepçe yavaşça sıkın. </li><li> Şırınga tutucu pipet tutucu içine yerleştirin. </li><li> Pipet enjektör enjeksiyon enjektör içine yavaşça kaydırın. Piston ucu şırınganın deliğe doğrudan beslenen emin olun. Bir kez tam yerleştiğinden, pipet enjektör içine şırınga sahibinin kilitleme yaka vida. </li></ol> 4. 14 örnek hücre yükleme <ol><li> Tampon çalışan en az 50 ml örnek hücre yıkayın ve uzun Needled 2.5 ml cam şırınga kullanarak kalan herhangi bir sıvı kaldırmak. </li><li> Yavaş yavaş, temiz ve kuru uzun Needled 2.5 ml şırınga içine protein örnek solüsyon en az 1.8 ml çizin. Kabarcıkları tanıtmak için dikkatli olun. </li><li> Iğne örnek hücre dikkatlice yerleştirin ve hafifçe hücrenin altındaki dokunun. Ucu hafifçe kaldırın (~ 1 mm) ve hücre içine aşırı sıvı örnek hücrenin üst üstünde görünür oluncaya kadar yavaşça AAC (6 &#39;)-Ii çözüm enjekte. </li><li> Taşma sıvı kalıntıları sağlarken 1 cm iğne yavaşça kaldırın. Hızla geri çekilmesi ve Örnek hücresi içinde sıkışan kabarcıkları kaldırmak için çözüm küçük bir miktar (~ 0.25 ml) enjekte edilir. </li><li> Tüm çözüm taşması çıkarın. Bu örnek hücre içine hafifçe taşması kenarı boyunca iğne sürgülü elde edilir. Şırınga ucu bir çıkıntıya vuracaktır, bu, çalışan bir çözüm için arzu yüksekliği. Bu çizginin üstünde oturan tüm sıvı çıkarın. </li></ol> 5. Yükleme ve enjeksiyon şırınga vadede 14 başlatılması <ol><li> Plastik tüp yükleme şırınga dolgu portuna takın. </li><li> Alt-üst dolgu liman piston ucu. </li><li> Pipet tutucu altındaki tüp dolum pipet AcCoA çözüm yerleştirin. Şırınga ucu dolum tüpü alt temas etmemelidir. </li><li> Küçük bir miktar yükleme şırınga tüp girene kadar yavaşça enjektöre çözüm çizin. </li><li> Piston ucu tıklatın ve Kapat Dolgu Port düğmesine düşürerek doldurma portu kapatın. Temizle ve dolum, herhangi bir hava kabarcığı kaldırmak için Temizle-&gt; Yedek düğmesine 3 kez tıklayarak yükleme sırasında tuzak haline olabilir. </li><li> Pipet tutucu dolum tüpü çıkarın ve şırınga ucu hafifçe silin. </li><li> Örnek hücre içine pipet montaj ve hafifçe daha düşük şırıngaya çekin. Şırınga kolayca viraj gibi yavaş yavaş devam edin, büyük bir titizlikle ihtiyaç vardır. Şırınga tamamen yaka kilitleme baz bastırarak takılı olduğundan emin olun. </li><li> İstenen çalışan sıcaklığı (20 ° C Bu durumda) ayarlayın ve (bu durumda 20μcal/sec) beklenen maksimum enjeksiyon ısı akışı biraz daha fazla bir referans güç seçin. </li><li> İstenen enjeksiyon hacimleri ve gecikmeler Programı. (Bu durumda, 28 enjeksiyonları istihdam edilmiştir. Ilk enjeksiyonu 60 sn gecikme ile 2 mcL hacmi vardı. Sonraki tüm enjeksiyonları ile 10 330 ler gecikmeler mcL hacimleri vardı.) </li></ol> 6. Müteakip çalışır <ol><li> Sonraki tüm çalışır 2,4,8,16 bir faktör AAC (6 &#39;)-Ii ve AcCoA konsantrasyonları azaltırken, 2-5 arasındaki adımları tekrarlayın ve 32. </li></ol> 7. Veri Analizi <ol><li> Küresel bağlayıcı parametreleri tek bir set tüm izotermleri uyan uyan yordamı kullanın, daha önce 10 tarif . </li></ol> 8. Temsilcisi Sonuçlar Temsilcisi veriler Şekil 1&#39;de gösterilmiştir. Izotermleri şekiller konsantrasyon farklı olmalıdır. Üstü için keskin geçişleri bekleniyor c-değerleri (yani yüksek protein ve ligand konsantrasyonları) (Şekil 2). AAC (6 &#39;)-Ii durumda, iki site sıralı modeli açıklayan iki takım, ayarlanabilir stoichiometries ile aynı, bağımsız sitelerin birden daha iyi bir uyum sağlar. <img src="/files/ftp_upload/2529/2529fig1.jpg" alt="Şekil 1" /> Şekil 1, AAC (6 &#39;)-Ii (192 mcM) AcCoA titrasyonu (3.86 mM) tarafından üretilen İzotermleri . A) Ham ITC iz. B) 2-sitesi sıralı uyum (bağlama parametreleri (kareler) belirlemek için kullanılan Entegre değerleri -). <img src="/files/ftp_upload/2529/2529fig2.jpg" alt="Şekil 2" /> AcCoA Şekil 2 ITC izotermleri değişik konsantrasyonlarda AAC (6 &#39;)-Ii içine titre. Deneysel veriler (açık çevrelerin), 2-setleri-siteleri bağımsız model (kesikli kırmızı) ve 2-site sıralı modeli (düz mavi) için uygun. 2-site sequentional modeli açıkça daha iyi bir genel anlaşma verir. Istihdam konsantrasyonları), 6 mcM, 0,25 mM, B) 12 mcM, 0.25 mM, C) 24 mcM, 0.5 mM D) 48 mcM, 1.0 mM, E) 96 mcM, 1.9 mM ve F) 196 mcM sırasıyla AAC (6 &#39;)-Ii ve AcCoA için 3.86 mM.

<ol>
	<li>Cliff, M. J., Ladbury, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+year+2002+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2002 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 16, 383-391 (2003).</li><li>Cliff, M. J., Gutierrez, A., Ladbury, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+year+2003+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2003 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 17, 513-523 (2004).</li><li>Ababou, A., Ladbury, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survey+of+the+year+2004:+literature+on+applications+of+isothermal+titration+calorimetry.">Survey of the year 2004: literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 19, 79-89 (2006).</li><li>Ababou, A., Ladbury, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survey+of+the+year+2005:+literature+on+applications+of+isothermal+titration+calorimetry.">Survey of the year 2005: literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 20, 4-14 (2007).</li><li>Okhrimenko, O. k. s. a. n. a., J, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+year+2006+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2006 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 21, 1-19 (2008).</li><li>Bjelic, S., Jelesarov, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+year+2007+literature+on+applications+of+isothermal+titration+calorimetry.">A survey of the year 2007 literature on applications of isothermal titration calorimetry.</a> Journal of Molecular Recognition. 21, 289-312 (2008).</li><li>Leavitt, S., Freire, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+protein+binding+energetics+by+isothermal+titration+calorimetry.">Direct measurement of protein binding energetics by isothermal titration calorimetry.</a> Current Opinion in Structural Biology. 11, 560-566 (2001).</li><li>Wiseman, T., Williston, S., Brandts, J. F., Lin, L. -. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+measurement+of+binding+constants+and+heats+of+binding+using+a+new+titration+calorimeter.">Rapid measurement of binding constants and heats of binding using a new titration calorimeter.</a> Analytical Biochemistry. 179, 131-137 (1989).</li><li>Capaldi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+X-Ray+Structure+of+Zebrafish+(Danio+rerio)+Ileal+Bile+Acid-Binding+Protein+Reveals+the+Presence+of+Binding+Sites+on+the+Surface+of+the+Protein+Molecule.">The X-Ray Structure of Zebrafish (Danio rerio) Ileal Bile Acid-Binding Protein Reveals the Presence of Binding Sites on the Surface of the Protein Molecule.</a> Journal of Molecular Biology. 385, 99-116 (2009).</li><li>Freiburger, L. A., Auclair, K., Mittermaier, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidating+Protein+Binding+Mechanisms+by+Variable-c+ITC.">Elucidating Protein Binding Mechanisms by Variable-c ITC.</a> ChemBioChem. 10, 2871-2873 (2009).</li><li>Wybenga-Groot, L. E., Draker, K. -. a., Wright, G. D., Berghuis, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+an+aminoglycoside+6'-N-acetyltransferase:+defining+the+GCN5-related+N-acetyltransferase+superfamily+fold.">Crystal structure of an aminoglycoside 6'-N-acetyltransferase: defining the GCN5-related N-acetyltransferase superfamily fold.</a> Structure. 7, 497-507 (1999).</li><li>Draker, K., Northrop, D. B., Wright, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+Mechanism+of+the+GCN5-Related+Chromosomal+Aminoglycoside+Acetyltransferase+AAC(6')-Ii+from+Enterococcus+faecium:+Evidence+of+Dimer+Subunit+Cooperativity.">Kinetic Mechanism of the GCN5-Related Chromosomal Aminoglycoside Acetyltransferase AAC(6')-Ii from Enterococcus faecium: Evidence of Dimer Subunit Cooperativity.</a> Biochemistry. 42, 6565-6574 (2003).</li><li>Wright, G. D., Ladak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+and+characterization+of+the+chromosomal+aminoglycoside+6'-N-acetyltransferase+from+Enterococcus+faecium.">Overexpression and characterization of the chromosomal aminoglycoside 6'-N-acetyltransferase from Enterococcus faecium.</a> Antimicrob. Agents Chemother. 41, 956-960 (1997).</li><li>MicroCal. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ITC Data Analysis in Origin. , (2004).</li></ol>

Bu değişken c uydurma analitik bir kısmı daha önce ayrıntılı 10 tarif edilmiştir . İşte biz, bu yaklaşım için uygun değişken-c veri setleri toplama pratik yönlerini rapor. Bu protein ve ligand örnekleri aynı stok çözümleri çizilmiş olması esastır. Bu nedenle yeterli stok solüsyonu deneylerin tüm seriyi tamamlamak için başlangıçta hazır olduğunu önemlidir. Bu oranı AAC (6 &#39;)-Ii sağlar ve AcCoA tüm deneyler arasında sürekli ve rastgele bir örnek-örnek bazında konsa...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Acetyl coenzyme A (AcCoA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A2056</td>
<td>&nbsp;</td>
<tr>
<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>7365-45-9</td>
<td>&nbsp;</td>
<tr>
<td>ethylenediaminetetraacetic acid (EDTA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>431788</td>
<td>&nbsp;</td>
<tr>
<td>Spectra/Por 2 Dialysis Tubing</td>
<td>&nbsp;</td>
<td>Spectrum Labs</td>
<td>132678</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Syringe Filter (0.2 &mu;m)</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>281445-477</td>
<td>&nbsp;</td>
<tr>
<td>Cellulos Nitrate Membrane Filters (0.45 &mu;m)</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>7184-004</td>
<td>&nbsp;</td>
<tr>
<td>VP-ITC</td>
<td>&nbsp;</td>
<td>MicroCal</td>
<td>VP-ITC</td>
<td>Microcalorimeter used for measurements</td>
</tr>
<tr>
<td>ThermoVac</td>
<td>&nbsp;</td>
<td>MicroCal</td>
<td>USB Thermo Vac</td>
<td>Temperature Controlled Degassing Station</td>
</tr>		</tbody>
		</table>
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collecting variable-concentration isothermal titration calorimetry datasets in order to determine binding mechanisms

Izotermal titrasyon kalorimetri (ITC) yaygın bir ana makromolekül bir ligand bağlama ile ilgili termodinamik parametreleri belirlemek için kullanılır. ITC konak / ligand etkileşimleri çalışmak için ortak spektroskopik yaklaşımlar üzerinde bazı avantajları vardır. Örneğin, serbest ya da iki bileşenden etkileşime girdiğinde emilir ısı doğrudan ölçülür ve herhangi bir eksojen gazetecilere gerektirmez. Bu nedenle bağlayıcı entalpi ve dernek sabiti (Ka), ITC verileri doğrudan elde edilen ve entropik katkısını hesaplamak için kullanılabilir. Ayrıca, izoterm şekli c-değeri ve katılan mekanistik model bağlıdır. C-değeri olarak tanımlanır c = n [P] TKA, [P] t protein konsantrasyonu ve n ev sahibi içinde ligand bağlayıcı sitelerinin sayısı. Pek çok durumda, belirli bir ligand için birden fazla bağlayıcı siteleri denk olmayan ve ITC, her bir bağlanma yeri için termodinamik bağlayıcı parametrelerinin karakterizasyonu sağlar. Ancak bu doğru bağlayıcı model gerektirir. Bu seçim, farklı modeller aynı deneysel verilere uygun eğer sorunlu olabilir. Biz daha önce bu sorunu c-değerleri çeşitli deneyler yaparak atlatılabilmişlerdir olabileceğini göstermiştir. C-değerleri farklı elde birden fazla izotermleri ayrı model aynı anda bir uyum vardır. Doğru model sonraki tüm değişken c veri kümesi arasında uyum iyiliği göre tespit edilir. Bu süreç, aminoglikozid direnci neden olan enzimi aminoglikozid N-6&#39;-asetiltransferaz-Ii (AAC (6 &#39;)-Ii) burada uygulanır. Bizim metodoloji herhangi bir sistem için geçerli olmakla birlikte, bu stratejinin gerekliliği daha iyi allostery veya cooperativity gösteren bir makromolekül-ligand sistemi ile gösterdi ve farklı bağlama modelleri aynı veri aslında aynı uyan. Bildiğimiz kadarıyla, piyasada bulunan böyle bir sistem vardır. AAC (6 &#39;)-Ii, iki alt arasındaki cooperativity gösteren iki aktif siteleri içeren bir homo-dimer. Ancak tek bir c-değeri elde ITC verileri eşit derecede iyi iki-setleri-siteleri bağımsız model ve sıralı bir iki yerinde (kooperatif) modeli en az iki farklı modelleri uygun olabilir. Yukarıda açıklandığı gibi c-değeri değişen sayesinde, doğru bağlama modelini AAC (6 &#39;)-Ii iki site sıralı bağlama modelini olduğunu kurulmuştur. Burada, biz ITC deneyler yaparken değişken c analizler için uygun veri setleri elde etmek için atılması gereken adımları açıklar.

Watch this Scientific Journal Video about Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms at JoVE.com

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

ITC, ev sahibi bir ligand bağlama eğitimi için güçlü bir araçtır. Ancak karmaşık sistemler, çeşitli modeller eşit derecede iyi veriler uygun olabilir. Burada anlatılan yöntem kompleks sistemler için uygun bir bağlama modelini aydınlatmak ve ilgili termodinamik parametreleri ayıklamak için bir araç sağlar.

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 ...

collecting-variable-concentration-isothermal-titration-calorimetry

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Biology

12.7K Görüntüleme.  McGill University. ITC, ev sahibi bir ligand bağlama eğitimi için güçlü bir araçtır. Ancak karmaşık sistemler, çeşitli modeller eşit derecede iyi veriler uygun olabilir. Burada anlatılan yöntem kompleks sistemler için uygun bir bağlama modelini aydınlatmak ve ilgili termodinamik parametreleri ayıklamak için bir araç sağlar.

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

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

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds 

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. Collecting site-specific biochemical markers allows us to evaluate the relationship between surgical wound and serum levels; determine any associations between mediator release, pain and analgesic consumption; and evaluate the effect of systemic and peripheral drug administration on surgical wound biochemistry.
This methodology has been applied to healthy women undergoing elective cesarean delivery with spinal anesthesia. Wound exudate and serum mediators, in conjunction with pain scores and analgesics consumption were measured at 1, 6, 24, and 48 hours post-cesarean delivery. Biochemical mediators that were detected included IL-1&beta;, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNF&alpha;, INF&gamma;, G-CSF, GM-CSF, MCP-1 and MIP-1&beta;, nerve growth factor (NGF), prostaglandin E2 (PG-E2) and substance P. We found no correlations between wound and serum cytokines concentrations or time-release profiles (J Pain. 2008 Jul 9(7):650-7). This article describes and demonstrates the feasibility of collecting and assaying nociceptive and inflammatory mediators in surgical wounds at specific time points. The lack of significant correlations between serum and wound levels shows the importance of determining site-specific release if surgical wounds and localized pathologies are to be studied.

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds

A technique to collect and measure surgical wound biochemical mediators at specific time points.

Cerrahi Yaralar Nosiseptif ve İnflamatuar Arabulucular Toplama ve Ölçüm

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. ...

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

Proteaz Aktivitesi belirleme Proteaz Floresan Algılama Kit kullanın

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR-Klip - RNA Cilt Proteinler Transcriptome geniş Cilt Siteleri Belirlemek Amacıyla Bir Yöntem

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.

A general protocol for the use of isothermal titration calorimetry to monitor the binding thermodynamics for biological systems with moderate binding affinities is presented.

Makromolekül-ligand Affinity Ölçüm İzotermal Titrasyon Kalorimetre

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction.  In biological ...

Culturing Primary Rat Inner Medullary Collecting Duct Cells

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion.
Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories.

Arginine-vasopressin (AVP) controls fine-tuning of body water homeostasis through facilitating water reabsorption by renal principal cells. Here, we present a protocol for the cultivation of primary rat inner medullary collecting duct cells suitable for the elucidation of molecular mechanisms underlying AVP-mediated water reabsorption.

Kanal Hücreleri Toplama kültür İlköğretim Rat İç Medüller

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP ...

Biochemical Titration of Glycogen In vitro

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 metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

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 ...

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is achieved by reverse transcription of the RNA of interest using specific fluorescently labeled primers and subsequent analysis of the resulting cDNA fragments by denaturing polyacrylamide gel electrophoresis. Simultaneously, a traditional Sanger sequencing reaction is run on the gel to map the ends of the cDNA fragments to their exact corresponding bases. In contrast to 5&#39;-RACE (Rapid Amplification of cDNA Ends), where the product must be cloned and multiple candidates sequenced, the bulk of cDNA fragments generated by primer extension can be simultaneously detected in one gel run. In addition, the whole procedure (from reverse transcription to final analysis of the results) can be completed in one working day. By using fluorescently labeled primers, the use of hazardous radioactive isotope labeled reagents can be avoided and processing times are reduced as products can be detected during the electrophoresis procedure.
In the following protocol, we describe an in vivo fluorescent primer extension method to reliably and rapidly detect the 5&#39; ends of RNAs to deduce transcriptional starting points and RNA processing sites (e.g., by toxin-antitoxin system components) in S. aureus, E. coli and other bacteria.

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.

Floresan Bazlı Astar Uzatma Tekniği Transkripsiyonal Başlangıç ​​Noktalarını ve RNases yarılması Siteleri belirleme<em&gt; In Vivo</em

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Ekstraselüler Asit Üretim Ölçüm ve Analiz Glikolitik Hızı belirleme

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.