Ce travail a été soutenu par les Instituts canadiens de recherche en santé du Canada (IRSC), en sciences naturelles et en génie (CRSNG), et une bourse d&#39;études de formation des IRSC (pour LF). Nous remercions le professeur Gérard D. Wright (Université McMaster, Canada) pour l&#39;AAC (6)-Ii plasmide d&#39;expression.

1. Préparation des solutions mères <ol><li> Purifier la macromolécule d&#39;intérêt. (Dans ce cas, aminoglycoside N-acétyl-6&#39;-II (AAC6&#39;-II), est isolé comme rapporté ailleurs. 13) </li><li> Préparer 4 litres de tampon de dialyse. (Dans ce cas nous avons utilisé 25 mm 4 - (2-hydroxyéthyl)-1-pipérazineéthanesulfonique (HEPES, MW 238,3 g / mol), contenant 2 mM d&#39;acide éthylène diamine (EDTA, MW 292.2), à pH 7,5.) </li><li> Dialyser la protéine L&#39;AAC (6 &#39;)-II de l&#39;échantillon (5 ml à 400 uM) utilisés doivent être dialysées dans un tampon de dialyse (3 x 1,3 L). La solution de dialyse final est conservé pour rincer la machine et de diluer les échantillons. </li><li> Filtrer la solution de dialyse final à travers un filtre de 0,45 um de cellulose, et conservés à 4 ° C. Sera dénommé «tampon courant&quot; </li><li> Solution de protéines Filtrer à travers un filtre de 0,2 um seringue qui a été rincé avec un tampon d&#39;exécution. Mesurer la concentration finale de la protéine en utilisant un dosage standard (Bradford, Lowry, etc) ou absorbance UV. Magasin de la protéine afin de maximiser la stabilité à long terme. (Dans le cas de l&#39;AAC (6 &#39;)-II, ce moyen de stockage à 4 ° C. AAC (6&#39;)-II ne conservent leur activité après congélation et décongélation). </li><li> Préparer 200 pl de 25 mM de solution stock acétyl-coenzyme A (AcCoA, le ligand, MW 809.57) en dissolvant 4,0 mg dans la gestion de la mémoire tampon. Gel à -78 ° C jusqu&#39;au moment de servir. </li><li> Toutes les protéines et les échantillons de ligand doivent provenir du même stock de solutions, afin de minimiser échantillon aléatoire de l&#39;échantillon à des fluctuations dans la concentration. Un seul facteur de correction pour la concentration de protéines dans l&#39;ensemble de variables-c données est ajusté dans l&#39;analyse. 10 </li></ol> 2. Préparation des échantillons CCI <ol><li> Diluer la solution enzymatique à un c-valeur de 64 avec un volume final de 2 ml. (Dans le cas de l&#39;AAC (6 &#39;)-II, ce qui correspond à 192 pM.) </li><li> Vite dégeler la solution stock ligand (AcCoA) dans l&#39;eau glacée. </li><li> Préparer 0,5 mL d&#39;une solution AcCoA 10 fois plus concentré, puis le nombre de sites de liaison sur la protéine, dans ce cas, 4 mM, en diluant 80 pl de la solution stock dans 420 uL AcCoA de courir tampon. Transférer dans un tube de la pipette de remplissage. Revenir rapidement la solution stock AcCoA à -78 ° C. </li><li> Degas la protéine et des solutions sous vide pendant 5 min à une température de 1 ° C en dessous de la température désirée de fonctionnement (19 ° C). </li></ol> 3. Mise en place de la seringue 14 <ol><li> Insérer la seringue d&#39;injection à travers porte-seringue jusqu&#39;à ce que l&#39;attache de la seringue pré-monté est à la même hauteur que le titulaire. </li><li> Feed the bride deuxième seringue au cours de la seringue jusqu&#39;à ce qu&#39;il soit fermement pressé contre le fond du porte-seringue. Serrer doucement la pince fournie avec les 0.050 &quot;entraînement hexagonal à bille. </li><li> Porte-seringue place dans le porte-pipette. </li><li> Faites glisser doucement l&#39;injecteur pipette dans la seringue. Assurez-vous que la pointe de piston est alimenté directement dans le trou de la seringue. Une fois entièrement inséré, visser la bague de blocage du titulaire dans l&#39;injecteur pipette. </li></ol> 4. Chargement de la cellule 14 de l&#39;échantillon <ol><li> Laver l&#39;échantillon de cellules avec un minimum de 50 ml de tampon de course, et enlever toute trace de liquide en utilisant un long aiguilleté 2,5 ml seringue en verre. </li><li> Lentement dessiner un minimum de 1,8 ml de solution échantillon de protéine dans le propre et sec à long aiguilleté 2,5 ml seringue. Attention à ne pas introduire de bulles. </li><li> Insérez soigneusement l&#39;aiguille dans la cellule de l&#39;échantillon et doucement toucher le fond de la cellule. Soulever la pointe légèrement (~ 1 mm) et d&#39;injecter délicatement AAC (6 &#39;)-II de solution dans la cellule jusqu&#39;à ce que l&#39;excès de liquide est visible au-dessus du haut de la cellule de l&#39;échantillon. </li><li> Soulevez lentement l&#39;aiguille d&#39;environ 1 cm tout en assurant reste liquide dans le débordement. Retirer rapidement et injecter une petite quantité de solution (~ 0,25 ml) pour éliminer les bulles emprisonnées dans la cellule d&#39;échantillon. </li><li> Retirez tous les débordement de solution. Ce résultat est obtenu en faisant délicatement l&#39;aiguille le long côté de déborder dans la cellule d&#39;échantillon. Le bout de la seringue va frapper une corniche, c&#39;est la hauteur désir de la solution fonctionne. Retirer tout le liquide qui se trouve au-dessus de cette ligne. </li></ol> 5. Chargement seringue d&#39;injection et d&#39;initier course de 14 <ol><li> Fixer la seringue tube en plastique de chargement dans le port de remplissage. </li><li> Pointe de la Basse piston pour le haut de l&#39;orifice de remplissage. </li><li> Placer la solution dans le tube AcCoA pipette de remplissage à bas de la porte-pipette. Le bout de la seringue ne doit pas toucher le fond du tube de remplissage. </li><li> Aspirer lentement la solution dans la seringue jusqu&#39;à ce qu&#39;une petite quantité pénètre dans le tube de la seringue de chargement. </li><li> Fermer l&#39;orifice de remplissage en abaissant le piston et cliquez sur le bouton Fermer l&#39;orifice de remplissage. Purge et recharge, en cliquant sur la Purge-&gt; bouton Recharge, trois fois pour éliminer les bulles d&#39;air qui peuvent avoir été piégés pendant le chargement. </li><li> Retirez le tube de remplissage du porte-pipette et essuyez délicatement l&#39;embout de la seringue. </li><li> Prenez la pipette et l&#39;assemblage de seringues abaisser doucement dans la cellule de l&#39;échantillon. Procéder lentement que la seringue peut facilement plier, donc le plus grand soin est nécessaire. Assurer la seringue est complètement inséré en appuyant sur la base du blocage col. </li><li> Régler la température désirée de fonctionnement (dans ce cas 20 ° C), et sélectionnez une puissance de référence qui est légèrement supérieur au flux de chaleur d&#39;injection maximale prévue (dans ce cas, 20μcal/sec). </li><li> Programme des volumes d&#39;injection désiré et les retards. (Dans ce cas, 28 injections ont été employées. La première injection a un volume de 2 pl avec un retard de 60 s. Toutes les injections suivantes ont des volumes de 10 ul avec des retards 330 s). </li></ol> 6. Des exécutions ultérieures <ol><li> Dans tous les exécutions ultérieures répétez les étapes 2-5, tout en diminuant les AAC (6 &#39;)-II et les concentrations AcCoA par un facteur de 2,4,8,16 et 32. </li></ol> 7. Analyse des données <ol><li> Utilisez une procédure d&#39;ajustement qui correspond globalement l&#39;ensemble des isothermes d&#39;un ensemble unique de paramètres de liaison, comme décrit précédemment 10. </li></ol> 8. Les résultats représentatifs Des données représentatives sont présentées dans la figure 1. Les formes des isothermes devrait varier avec la concentration. Sharper transitions sont attendus pour plus c-valeurs (ie protéines élevé et les concentrations de ligand) (figure 2). Dans le cas de l&#39;AAC (6 &#39;)-II, le modèle à deux sites séquentielle donne un meilleur choix que celui décrivant deux ensembles de produits identiques, avec des sites indépendants stoechiométries réglable. <img src="/files/ftp_upload/2529/2529fig1.jpg" alt="Figure 1" /> Isothermes Figure 1. Produite par un titrage des AcCoA (3,86 mM) en AAC (6 &#39;)-II (192 pM). A) trace premières CCI. B) des valeurs intégrées utilisées pour déterminer des paramètres obligatoires (places) avec un ajustement 2 place séquentielle (-). <img src="/files/ftp_upload/2529/2529fig2.jpg" alt="Figure 2" /> Figure 2. Isothermes CCI pour AcCoA titré en AAC (6 &#39;)-II à des concentrations variables. Les données expérimentales (cercles ouverts) ont été adaptés à un modèle 2-ensembles-de-sites indépendants (magenta pointillés) et un modèle 2-place séquentielle (solide bleu). Le modèle 2-site sequentional donne clairement un accord globalement meilleure. Les concentrations utilisées étaient de type A) 6 uM, 0,25 mm, B) 12 uM, 0,25 mm, C) 24 uM, 0,5 mM, D) 48 uM, 1,0 mM, E) 96 uM, 1,9 mM, et F) 196 uM, 3,86 mM, pour AAC (6 &#39;)-II et AcCoA respectivement.

<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>

Cette partie analytique de variable c montage a été précédemment décrit en détail 10. Nous rapportons ici les aspects pratiques de la collecte variable c ensembles de données adapté à cette approche. Il est essentiel que toutes les protéines et les échantillons de ligand sont tirées des solutions même stock. Il est donc important que la solution stock suffisant est préparé initialement pour compléter la série entière d&#39;expériences. Cela garantit le ratio de l&#39;AAC (6 &#39;)-II et AcC...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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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>
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collecting variable-concentration isothermal titration calorimetry datasets in order to determine binding mechanisms

Calorimétrie de titrage isotherme (ITC) est couramment utilisé pour déterminer les paramètres thermodynamiques associés à la liaison d&#39;un ligand à une macromolécule hôte. ITC a certains avantages sur des approches communes pour étudier par spectroscopie d&#39;hôte / ligand interactions. Par exemple, la chaleur dégagée ou absorbée lorsque les deux composants interagissent est directement mesurée et ne nécessite aucune journalistes exogènes. Ainsi, l&#39;enthalpie de liaison et la constante d&#39;association (Ka) sont obtenues directement à partir de données CCI, et peut être utilisé pour calculer la contribution entropique. Par ailleurs, la forme de l&#39;isotherme est dépendante de la Valeur-C et le modèle mécaniste impliqués. La Valeur-C est défini comme c = n [P] TKA, où [P] l est la concentration en protéines, et n est le nombre de sites de liaison au ligand au sein de l&#39;hôte. Dans de nombreux cas, plusieurs sites de liaison pour un ligand donné ne sont pas équivalents et l&#39;ITC permet la caractérisation des paramètres thermodynamiques de liaison pour chaque site individuel contraignant. Cela exige cependant que le modèle correct contraignante être utilisé. Ce choix peut être problématique si les différents modèles peuvent tenir les mêmes données expérimentales. Nous avons précédemment montré que ce problème peut être contourné en réalisant des expériences dans plusieurs C-valeurs. Les isothermes de multiples obtenues à différentes valeurs de c-sont aptes simultanément à des modèles séparés. Le modèle correct est ensuite identifié sur la base des qualité de l&#39;ajustement sur l&#39;ensemble de variables-c données. Ce processus est appliqué ici à l&#39;aminoglycoside résistance provoquant aminoglycoside enzyme N-acétyl-6&#39;-II (AAC (6 &#39;)-II). Bien que notre méthodologie est applicable à tout système, la nécessité de cette stratégie est mieux démontrée avec un système ligand-macromolécule montrant allostérie ou coopérativité, et quand différents modèles de fixation s&#39;adapte fournissent essentiellement identiques aux mêmes données. À notre connaissance, il n&#39;existe pas de tels systèmes disponibles dans le commerce. AAC (6 &#39;)-II, est un homo-dimère contenant deux sites actifs, en montrant la coopérativité entre les deux sous-unités. Toutefois, les données obtenues à l&#39;ITC d&#39;un seul c-valeur peut être adapté aussi bien à au moins deux modèles différents d&#39;un modèle à deux ensembles de sites indépendants et une à deux sites séquentielle (coopérative) modèle. Grâce à varier le C-valeur comme expliqué ci-dessus, il a été établi que le modèle correct contraignante pour AAC (6 &#39;)-II est un site de deux modèle séquentiel contraignant. Nous décrivons ici les étapes qui doivent être prises lors de la réalisation d&#39;expériences CCI en vue d&#39;obtenir des ensembles de données adapté à la variable c-analyses.

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 est un outil puissant pour étudier la liaison d&#39;un ligand à son hôte. Dans les systèmes complexes cependant, plusieurs modèles peuvent ajuster les données aussi bien. La méthode décrite ici fournit un moyen d&#39;élucider le modèle approprié contraignante pour les systèmes complexes et d&#39;extraire les paramètres thermodynamiques correspondants.

Collecte variable de concentration isotherme ensembles de données de titrage calorimétrique afin de déterminer des mécanismes de liaison

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

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12.7K vues.  McGill University. ITC est un outil puissant pour étudier la liaison d'un ligand à son hôte. Dans les systèmes complexes cependant, plusieurs modèles peuvent ajuster les données aussi bien. La méthode décrite ici fournit un moyen d'élucider le modèle approprié contraignante pour les systèmes complexes et d'extraire les paramètres thermodynamiques correspondants.

Vidéo: 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.

Le titrage de coronavirus humains en utilisant un dosage immunoperoxydase

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

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Biologie

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.

Recueillir et mesurer les médiateurs nociceptifs et inflammatoires dans les plaies chirurgicales

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.

L&#39;utilisation du kit de détection de protéase fluorescent pour déterminer l&#39;activité de protéase

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-Clip - Une méthode pour identifier transcriptome à l&#39;échelle des sites de liaison des protéines liant l&#39;ARN

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.

Calorimétrie titration isotherme pour Mesure macromolécules-ligand d&#39;affinité

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.

Culture primaire Rat collecteur médullaire interne les cellules des canaux

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.

Titration biochimique de glycogène<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.

Fluorescence Basé Technique d&#39;extension d&#39;amorce pour déterminer transcription Points de départ et des sites de clivage de RNases<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.

Mesure et analyse de la production extracellulaire acide pour Déterminer glycolytique Taux

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.