Questo lavoro è stato sostenuto dal Canadian Institutes of Health Research (CIHR), National Science and Engineering Research Council (NSERC), e una formazione CIHR borsa di studio (di LF). Ringraziamo il Prof. Gerard D. Wright (McMaster University, Canada) per la CAA (6)-Ii plasmide di espressione.

1. Preparazione di soluzioni di riserva <ol><li> Purificare la macromolecola di interesse. (In questo caso, aminoglicosidi N-6&#39;-acetiltransferasi-Ii (AAC6&#39;-II), è isolato come riportato altrove. 13) </li><li> Preparare 4 litri di buffer di dialisi. (In questo caso abbiamo utilizzato 25 mm 4 - (2-idrossietil)-1-piperazineethanesulfonic acido (HEPES, MW 238,3 g / mol), contenente 2 mM acido etilendiamminotetraacetico (EDTA, 292,2 MW), a pH 7.5.) </li><li> Dializzare la proteina La (6 &#39;)-Ii AAC campione (5 ml a 400 mM) utilizzati devono essere dializzati nel buffer di dialisi (3 x 1,3 L). La soluzione di dialisi finale è tenuto a lavare la macchina e per diluire campioni. </li><li> Filtrare la soluzione finale dialitica attraverso un filtro da 0,45 micron di cellulosa, e conservato a 4 ° C. Sarà denominato &#39;tampone di corsa&#39; </li><li> Proteine ​​filtro soluzione attraverso un filtro 0,2 micron siringa che è stato sciacquato accuratamente con tampone di corsa. Misurare la concentrazione finale della proteina usando un test standard (Bradford, Lowry, ecc) o assorbimento UV. Conservare la proteina per ottimizzare stabilità a lungo termine. (Nel caso di AAC (6 &#39;)-Ii, questo significa conservazione a 4 ° C. AAC (6&#39;)-Ii non mantiene l&#39;attività dopo il congelamento e scongelamento). </li><li> Preparare 200μL soluzione madre di 25 mM di acetil coenzima A (ACCOA, il ligando, 809,57 MW), sciogliendo 4,0 mg nella gestione del buffer. Congelare a -78 ° C fino al momento dell&#39;uso. </li><li> Tutte le proteine ​​e campioni ligando deve provenire dalla soluzione madre stessa, per ridurre al minimo casuale campione a campione le fluttuazioni nella concentrazione. Un fattore di correzione unico per la concentrazione di proteine ​​su tutta la variabile-c set di dati viene regolata nell&#39;analisi 10. </li></ol> 2. Preparazione campioni ITC <ol><li> Diluire la soluzione enzimatica di un c-valore di 64 con un volume finale di 2 ml. (Nel caso di AAC (6 &#39;)-Ii, ciò corrisponde a 192 micron.) </li><li> Rapidamente il disgelo (ACCOA) soluzione di legante magazzino in acqua ghiacciata. </li><li> Preparare 0,5 mL di una soluzione ACCOA 10 volte più concentrato quindi il numero di siti di legame della proteina, in questo caso 4 mm, diluendo 80 ml di soluzione madre ACCOA in 420 ml di buffer di esecuzione. Trasferire in un tubo pipetta di riempimento. Tornare rapidamente alla soluzione madre ACCOA a -78 ° C. </li><li> Degas le proteine ​​e le soluzioni sotto vuoto per 5 minuti ad una temperatura 1 ° C al di sotto della temperatura desiderata in esecuzione (19 ° C). </li></ol> 3. Impostazione della siringa 14 <ol><li> Inserire la siringa attraverso titolare siringa fino a quando il morsetto siringa pre-montato è alla stessa altezza del titolare. </li><li> Alimentare il secondo morsetto siringa sopra la siringa d&#39;iniezione fino a quando è saldamente premuto contro la parte inferiore del titolare siringa. Stringere la fascetta con l&#39;apposita 0,050 &quot;Drive Hex Point Ball. </li><li> Luogo titolare siringa nel supporto pipetta. </li><li> Far scorrere delicatamente l&#39;iniettore pipetta nella siringa d&#39;iniezione. Assicurarsi che la punta pistone è alimentato direttamente nel buco della siringa. Una volta completamente inserito, avvitare il collare di bloccaggio del titolare siringa nel iniettore pipetta. </li></ol> 4. Caricamento della cella campione 14 <ol><li> Lavare le cellule campione con un minimo di 50 ml di tampone in esecuzione e rimuovere eventuali residui di liquido utilizzando un lungo ago, siringa 2,5 ml di vetro. </li><li> Lentamente disegnare un minimo di 1,8 ml di soluzione campione di proteine ​​nel pulito ed asciutto lungo agugliato siringa 2,5 ml. Fare attenzione a non introdurre eventuali bolle. </li><li> Inserire con cautela l&#39;ago nella cella del campione e delicatamente toccare il fondo della cella. Alzare punta leggermente (~ 1 mm), iniettare delicatamente AAC (6 &#39;)-Ii soluzione nella cella fino a quando il liquido in eccesso è visibile sopra la parte superiore della cella campione. </li><li> Sollevare lentamente l&#39;ago di circa 1 cm, garantendo nel contempo rimane liquido nel overflow. Ritirare rapidamente e iniettare una piccola quantità di soluzione (~ 0,25 ml) per rimuovere eventuali bolle d&#39;aria intrappolate nella cella campione. </li><li> Rimuovere tutti i trabocco soluzione. Ciò si ottiene facendo scorrere delicatamente l&#39;ago lungo il lato di overflow nella cella del campione. La punta della siringa ha colpito una sporgenza, questa è l&#39;altezza desiderio per la soluzione in esecuzione. Rimuovere tutto il liquido che si trova sopra di questa linea. </li></ol> 5. Caricamento siringa e l&#39;avvio di eseguire 14 <ol><li> Attaccare la siringa di plastica tubo di carico in porta di riempimento. </li><li> Stantuffo punta inferiore alla parte superiore della porta di riempimento. </li><li> Posto la soluzione ACCOA nel tubo pipetta riempimento nella parte inferiore del titolare pipetta. La punta della siringa non deve toccare il fondo del tubo di riempimento. </li><li> Lentamente disegnare la soluzione nella siringa fino a quando una piccola quantità entra nel tubo della siringa di carico. </li><li> Chiudere la porta di riempire abbassando la punta del pistone e cliccare sul pulsante Chiudi Porto Fill. Spurgo e riempimento, cliccando sul Purga-&gt; pulsante Refill, 3 volte per rimuovere eventuali bolle d&#39;aria che possono essere stati intrappolati durante il caricamento. </li><li> Rimuovere il tubo di riempimento da parte del titolare pipetta e pulire delicatamente la punta della siringa. </li><li> Prendete il montaggio pipetta e siringa abbassare delicatamente nella cella del campione. Procedete lentamente la siringa può facilmente piegare, così grande cura è necessaria. Assicurarsi che la siringa sia completamente inserito premendo sulla base del collare di bloccaggio. </li><li> Impostare la temperatura desiderata in esecuzione (in questo caso 20 ° C), e selezionare una potenza di riferimento che è leggermente superiore alla portata massima di iniezione di calore prevista (in questo caso 20μcal/sec). </li><li> Programmare il volume di iniezione desiderato e ritardi. (In questo caso, 28 iniezioni sono state impiegate. La prima iniezione aveva un volume di 2 microlitri con un ritardo di 60 s. Tutte le iniezioni successive avevano volumi di 10 L con ritardi di 330 s). </li></ol> 6. Esecuzioni successive <ol><li> In tutte le esecuzioni successive ripetere i passaggi 2-5, mentre diminuisce AAC (6 &#39;)-Ii e concentrazioni ACCOA di un fattore di 2,4,8,16 e 32. </li></ol> 7. Analisi dei dati <ol><li> Utilizzare una procedura di montaggio che si inserisce a livello globale di tutte le isoterme ad un unico insieme di parametri di associazione, come descritto in precedenza 10. </li></ol> 8. Rappresentante Risultati Dati rappresentativi sono mostrati in Figura 1. Le forme delle isoterme deve variare con la concentrazione. Più nitide le transizioni sono attesi per una maggiore c-valori (ad esempio proteine ​​superiore e le concentrazioni di ligando) (Figura 2). Nel caso di AAC (6 &#39;)-Ii, i due-sito modelli sequenziale dà una misura migliore di quella che descrive due gruppi di uguali, siti indipendenti con stechiometrie regolabile. <img src="/files/ftp_upload/2529/2529fig1.jpg" alt="Figura 1" /> Isoterme Figura 1. Prodotta dalla titolazione di ACCOA (3,86 mM) in AAC (6 &#39;)-Ii (192 mM). A) traccia ITC Raw. B) I valori integrato utilizzato per determinare parametri di associazione (quadrati) con una vestibilità 2-site sequenziale (-). <img src="/files/ftp_upload/2529/2529fig2.jpg" alt="Figura 2" /> Figura 2. Isoterme ITC per ACCOA titolato in AAC (6 &#39;)-Ii a concentrazioni variabili. I dati sperimentali (cerchi aperti) erano idonei ad un modello 2-set-di-siti indipendenti (magenta tratteggiata) e un 2-sito modelli sequenziale (solido blu). Il 2-sito modelli sequentional dà chiaramente un accordo generale migliore. Le concentrazioni sono state impiegate A) 6 micron, 0,25 mm, B) 12 mM, 0,25 mM, C) 24 mM, 0,5 mM, D) 48 mM, 1,0 mM, E) 96 mM, 1,9 mm, ed F) 196 micron, 3,86 mm, per AAC (6 &#39;)-Ii e ACCOA rispettivamente.

<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>

Nessun conflitto di interessi dichiarati.

Questa porzione analitica di variabile-c raccordo è stato precedentemente descritto in dettaglio 10. Qui riportiamo gli aspetti pratici della raccolta di set di dati idonei per questo approccio variabile-c. E &#39;essenziale che tutte le proteine ​​e campioni ligando sono tratti dalle soluzioni stesso ceppo. Perciò è importante che la soluzione scorte sufficienti è preparato inizialmente per completare l&#39;intera serie di esperimenti. In questo modo il rapporto tra AAC (6 &#39;)-Ii e ACCOA è costan...

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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>
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collecting variable-concentration isothermal titration calorimetry datasets in order to determine binding mechanisms

Calorimetria isotermica di titolazione (ITC) è comunemente usato per determinare i parametri termodinamici associati al legame di un ligando ad una macromolecola host. ITC ha alcuni vantaggi rispetto ai comuni approcci spettroscopiche per lo studio di host / ligando interazioni. Per esempio, il calore prodotto o assorbito quando le due componenti interagiscono viene misurata direttamente e non richiede alcuna giornalisti esogeni. Così l&#39;entalpia vincolante e la costante di associazione (Ka) sono direttamente ottenute dai dati ITC, e può essere utilizzato per calcolare il contributo entropico. Inoltre, la forma della isoterma dipende dal c-valore e il modello meccanicistico coinvolti. Il c-valore è definito come c = n [P] PTG, dove [P] t è la concentrazione di proteine, e n è il numero di siti di legame all&#39;interno dell&#39;ospite. In molti casi, diversi siti di legame per un ligando dato non sono equivalenti e ITC permette la caratterizzazione dei parametri termodinamici vincolanti per ogni singolo sito vincolanti. Ciò richiede tuttavia che il modello corretto vincolante essere utilizzato. Questa scelta può essere problematico se diversi modelli può andare bene gli stessi dati sperimentali. Abbiamo già dimostrato che questo problema può essere aggirato effettuando esperimenti in diverse c-valori. Le isoterme più disponibili presso diversi c-i valori sono idonei contemporaneamente modelli separati. Il modello corretto è vicino identificato sulla base della bontà di adattamento su tutta la variabile-c dataset. Questo processo viene applicato qui per la aminoglicosidi resistenza che causano aminoglicosidi enzima N-6&#39;-acetiltransferasi-Ii (AAC (6 &#39;)-II). Anche se la nostra metodologia è applicabile a qualsiasi sistema, la necessità di questa strategia è meglio dimostrato con una macromolecola-ligando sistema che mostra allostery o cooperatività e, quando diversi modelli vincolanti fornire adatta sostanzialmente identica agli stessi dati. A nostra conoscenza, non esistono sistemi di questo tipo disponibile in commercio. AAC (6 &#39;)-II, è un homo-dimero contenente due siti attivi, che mostra cooperatività tra le due subunità. Tuttavia i dati ITC ottenuti ad un singolo c-valore può essere adatta altrettanto bene ad almeno due diversi modelli un modello a due-set-di-siti indipendenti e due siti sequenziale (cooperativa) modello. Attraverso variando il valore C come spiegato sopra, è stato stabilito che il modello corretto vincolante per AAC (6 &#39;)-II è un due siti modello sequenziale vincolanti. Qui, descriviamo i passi che devono essere presi durante l&#39;esecuzione di esperimenti ITC al fine di ottenere set di dati idonei per la variabile-c analisi.

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 è un potente strumento per studiare il legame di un ligando al suo ospite. Nei sistemi complessi tuttavia, alcuni modelli può montare il dati ugualmente bene. Il metodo qui descritto fornisce un mezzo per chiarire il modello appropriato vincolante per i sistemi complessi ed estrarre i parametri termodinamici corrispondenti.

Raccolta variabile concentrazione dataset Calorimetria isotermica di titolazione per determinare meccanismi vincolanti

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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Biology

12.7K Views.  McGill University. ITC è un potente strumento per studiare il legame di un ligando al suo ospite. Nei sistemi complessi tuttavia, alcuni modelli può montare il dati ugualmente bene. Il metodo qui descritto fornisce un mezzo per chiarire il modello appropriato vincolante per i sistemi complessi ed estrarre i parametri termodinamici corrispondenti.

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.

Titolazione del coronavirus umani utilizzando un test immunoperossidasi

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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Biologia

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.

Raccolta e misurazione nocicettivo mediatori infiammatori e nelle ferite chirurgiche

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;uso del kit di rilevamento a fluorescenza della proteasi per determinare l&#39;attività della proteasi

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 - un metodo per identificare trascrittoma livello i siti di legame delle proteine ​​RNA binding

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.

Calorimetria isotermica di titolazione di misura macromolecola-ligando di affinità

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.

Coltura primaria Rat midollare interna Raccolta cellule del dotto

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.

Titolazione biochimica di glicogeno<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.

Fluorescenza base tecnica Primer Extension determinare trascrizionali punti di partenza e siti di taglio di RNasi<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.

Misura e Analisi di extracellulare Acido prodotte per determinare glicolitica Tasso

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.