Diese Arbeit wurde von den Canadian Institutes of Health Research (CIHR), National Science and Engineering Research Council (NSERC) und eine CIHR Ausbildungsförderung Stipendium (LF) unterstützt. Wir danken Prof. Gerard D. Wright (McMaster University, Kanada) für den AAC (6)-Ii Expressionsplasmid.

1. Vorbereitung Stammlösungen <ol><li> Purify das Makromolekül von Interesse. (In diesem Fall ist Aminoglykosid N-6&#39;-Acetyltransferase-II (AAC6&#39;-II), isoliert wie an anderer Stelle berichtet. 13) </li><li> Bereiten Sie 4 Liter Dialyse-Puffer. (In diesem Fall haben wir 25 mM 4 - (2-hydroxyethyl)-1-piperazineethanesulfonic Säure (HEPES, MW 238,3 g / mol), enthaltend 2 mM Ethylendiamintetraessigsäure (EDTA, MW 292,2), bei pH 7,5.) </li><li> Dialysieren das Protein Das AAC (6 &#39;)-II Probe (5 mL bei 400 uM) verwendet in der Dialyse-Puffer (3 x 1,3 l) dialysiert werden müssen. Die endgültige Dialyselösung gehalten wird, um die Maschine spülen und die Proben zu verdünnen. </li><li> Filtern Sie die letzte Dialyse-Lösung durch einen 0,45 um Filter aus Zellulose, und bei 4 ° C. Will als &quot;Laufpuffer&quot; genannt werden </li><li> Filter Protein-Lösung durch einen 0,2 um Spritzenfilter, die gespült worden ist gründlich mit Laufpuffer. Messen Sie die Endkonzentration des Proteins mit einem Standard-Test (Bradford, Lowry, etc.) oder UV-Absorption. Shop des Proteins an langfristige Stabilität zu maximieren. (Im Falle von AAC (6 &#39;)-II bedeutet dies, Lagerung bei 4 ° C. AAC (6&#39;)-II nicht behalten Aktivität nach dem Einfrieren und Auftauen). </li><li> Bereiten Sie 200 ul 25 mM Stammlösung von Acetyl-Coenzym A (AcCoA, den Liganden, MW 809,57) durch Lösen von 4,0 mg in Laufpuffer. Einfrieren bei -78 ° C bis zur Verwendung. </li><li> Alle Protein und Ligand Proben müssen aus dem gleichen Stammlösungen stammen, zu Zufallsstichprobe-to-sample Schwankungen in der Konzentration zu minimieren. Eine einzige Korrekturfaktor für die Protein-Konzentration über die gesamte variable-c Datensatz wird in der Analyse angepasst. 10 </li></ol> 2. Vorbereitung ITC Proben <ol><li> Verdünnen Sie die Enzymlösung zu einer c-Wert von 64 mit einem Gesamtvolumen von 2 mL. (Im Falle von AAC (6 &#39;)-II, das entspricht zu 192 pM.) </li><li> Schnell Tauwetter der Ligand (AcCoA) Stammlösung in Eiswasser. </li><li> Bereiten Sie 0,5 ml einer Lösung AcCoA 10-mal stärker konzentriert sich die Anzahl der Bindungsstellen des Proteins, in diesem Fall 4 mM, die durch Verdünnen 80 ul der AcCoA Stammlösung in 420 ul der Laufpuffer. Transfer in eine Pipette Füllrohr. Schnell wieder die AcCoA Stammlösung zu -78 ° C. </li><li> Degas das Protein und Lösungen unter Vakuum für 5 min bei einer Temperatur 1 ° C unterhalb der gewünschten Betriebstemperatur (19 ° C). </li></ol> 3. Einrichten der Spritze 14 <ol><li> Legen Sie die Injektionsspritze durch Spritzenhalter bis die vormontierten Spritzenklemme ist auf der gleichen Höhe wie der Inhaber. </li><li> Vorschub der zweiten Spritzenklemme über die Injektionsspritze, bis es fest gegen den Boden der Spritze Halterung gedrückt. Leicht anziehen Klemme mit dem mitgelieferten 0,050 &quot;Ball Point Hex Drive. </li><li> Legen Spritze Halter in den Pipettenhalter. </li><li> Schieben Sie die Pipette Injektor in die Injektionsspritze. Achten Sie darauf, den Kolben Spitze direkt in das Loch der Spritze gefüttert. Einmal vollständig eingeführt ist, schrauben Sie den Spannring der Spritze Halter in die Pipette Injektor. </li></ol> 4. Lädt die Probe Zelle 14 <ol><li> Wash Probenzelle mit einem Minimum von 50 ml Laufpuffer und entfernen Sie alle verbleibenden Flüssigkeit unter Verwendung eines langnadligen 2,5 ml Glasspritze. </li><li> Langsam ziehen ein Minimum von 1,8 ml Protein Probenlösung in den sauberen und trockenen langnadligen 2,5 ml Spritze. Achten Sie darauf, keine Blasen einzuführen. </li><li> Vorsichtig Nadel in Probenzelle und sanft berühren den Boden der Zelle. Raise Spitze leicht (~ 1 mm) und leicht zu injizieren AAC (6 &#39;)-II-Lösung in die Zelle, bis die überschüssige Flüssigkeit sichtbar ist über dem oberen Ende der Probe Zelle. </li><li> Langsam heben Sie die Nadel etwa 1 cm und gleichzeitig Flüssigkeit bleibt in den Überlauf. Schnell zurückziehen und injizieren eine kleine Menge der Lösung (~ 0,25 ml) um eingeschlossene Luftblasen in der Probe Zelle zu entfernen. </li><li> Entfernen Sie alle Lösungen Überlauf. Dies wird durch leichtes Verschieben der Nadel an der Seite der Überlauf in die Probenzelle erreicht. Die Spitze der Spritze wird eine Leiste getroffen, das ist der Wunsch Höhe für das laufende Lösung. Entfernen Sie alle Flüssigkeit, die oberhalb dieser Linie sitzt. </li></ol> 5. Loading Injektionsspritze und Einleitung laufen 14 <ol><li> Befestigen Sie den Kunststoffschlauch loading Spritze in Einfüllöffnung. </li><li> Lower Kolbenspitze an die Spitze der Einfüllöffnung. </li><li> Legen Sie die AcCoA Lösung in die Pipette Füllrohr an der Unterseite des Pipettenhalter. Die Spitze der Spritze nicht berühren den Boden des Füllrohr. </li><li> Langsam ziehen Sie die Lösung in die Spritze, bis eine kleine Menge in die Röhre des Be-Spritze. </li><li> Schließen Sie die Einfüllöffnung durch eine Senkung der Kolbenspitze und klicken Sie auf die Schließen-Schaltfläche Port Fill. Purge und füllen, indem Sie auf die Purge-&gt; Refill-Taste, 3-mal um die Luftblasen zu entfernen, dass kann eingeschlossen werden während des Ladens haben. </li><li> Entfernen Sie den Füllschlauch vom Pipettenhalter und wischen Sie die Spitze der Spritze. </li><li> Nehmen Pipette Montage und senken Spritze in die Messküvette. Gehen Sie langsam, wie die Spritze leicht verbiegen kann, so große Sorgfalt erforderlich ist. Sicherstellen, dass die Spritze vollständig durch Drücken auf Basis der Spannring eingesetzt. </li><li> Stellen Sie die gewünschte Betriebstemperatur (in diesem Fall 20 ° C), und wählen Sie einen Hinweis Kraft, die etwas größer ist als der maximale Einspritzdruck Wärmestrom erwartet (in diesem Fall 20μcal/sec) ist. </li><li> Programmieren Sie die gewünschte Injektionsvolumina und Verzögerungen. (In diesem Fall wurden 28 Injektionen eingesetzt. Die erste Injektion hatte ein Volumen von 2 ul mit 60 s Verzögerung. Alle nachfolgenden Injektionen hatten Volumina von 10 ul mit 330 s verzögert.) </li></ol> 6. Nachfolgende Durchläufe <ol><li> In allen nachfolgenden Läufen wiederholen Sie die Schritte 2-5, während abnehmende AAC (6 &#39;)-II und AcCoA Konzentrationen mit einem Faktor von 2,4,8,16 und 32. </li></ol> 7. Data Analysis <ol><li> Verwenden Sie ein passendes Verfahren, das weltweit für alle der Isothermen zu einer einzigen Reihe von verbindlichen Parameter, wie zuvor beschrieben 10. </li></ol> 8. Repräsentative Ergebnisse Repräsentative Daten sind in Abbildung 1 dargestellt. Die Formen der Isothermen sollte mit der Konzentration variieren. Schärfere Übergänge sind für höhere erwartete c-Werte (dh höhere Protein und Ligand-Konzentrationen) (Abbildung 2). Im Falle von AAC (6 &#39;)-Ii, gibt den beiden vor Ort sequenziellen Modell eine bessere Passform als eine Beschreibung zwei Sätze identisch, unabhängig Sites mit verstellbaren Stöchiometrien. <img src="/files/ftp_upload/2529/2529fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Isothermen durch Titration AcCoA (3,86 mM) in AAC (6 &#39;)-II (192 nM) hergestellt. A) Raw ITC Spur. B) Integrierte Werte für die Ermittlung Bindungsparameter (Quadrate) mit einem 2-site sequentielle fit (verwendet -). <img src="/files/ftp_upload/2529/2529fig2.jpg" alt="Abbildung 2" /> Abbildung 2. ITC Isothermen für AcCoA in AAC (6 &#39;)-II in unterschiedlichen Konzentrationen titriert. Die experimentellen Daten (offene Kreise) wurden auf ein 2-sets-of-Sites unabhängigen Modell (gestrichelte magenta) und ein 2-site sequentielle Modell (durchgezogene blaue) passen. Die 2-site sequentional Modell eindeutig eine bessere allgemeine Einigung. Die eingesetzten Konzentrationen waren 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, und F) 196 uM, 3,86 mm, für AAC (6 &#39;)-II und AcCoA jeweils.

<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>

Diese analytische Teil der variablen c Montage wurde bereits im Detail 10 beschrieben. Hier berichten wir über die praktischen Aspekte des Sammelns variable-c-Datensätze für diesen Ansatz. Es ist wichtig, dass alle Protein und Ligand Proben aus dem gleichen Stammlösungen gezogen werden. Deshalb ist es wichtig, dass genügend Stammlösung wird zunächst die gesamte Reihe von Experimenten vollständig vorbereitet. Dadurch wird sichergestellt, das Verhältnis von AAC (6 &#39;)-II und AcCoA ist bei allen Expe...

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		<div>
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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

Isotherme Titrationskalorimetrie (ITC) wird häufig verwendet, um die thermodynamischen Parameter der Bindung eines Liganden an einen Host-Makromolekül verbunden zu bestimmen. ITC hat einige Vorteile gegenüber gängigen spektroskopischen Methoden zur Untersuchung von Host / Ligand-Wechselwirkungen. Zum Beispiel wird die freiwerdende Wärme oder absorbiert wird, wenn die beiden Komponenten interagieren direkt gemessen und erfordert keine exogenen Reporter. So ist die Bindungsenthalpie und der Assoziationskonstante (Ka) werden direkt von ITC-Daten erhalten, und kann verwendet werden, um die entropische Beitrag berechnen. Darüber hinaus ist die Form der Isotherme abhängig von der c-Wert und die mechanistischen Modell beteiligt. Die c-Wert ist definiert als c = n [P] TKA, wobei [P] t ist die Protein-Konzentration und n die Anzahl der Liganden-Bindungsstellen innerhalb der Host. In vielen Fällen sind mehrere Bindungsstellen für einen bestimmten Liganden nicht gleichwertig und ITC ermöglicht die Charakterisierung des thermodynamischen Bindungsparameter für jedes einzelne Bindungsstelle. Dies erfordert jedoch, dass die korrekte Bindung Modell verwendet werden. Diese Wahl kann problematisch sein, wenn verschiedene Modelle der gleichen experimentellen Daten passen. Wir haben bereits gezeigt, dass dieses Problem durch die Durchführung von Experimenten an mehreren c-Werte können nicht umgangen werden. Die multiple Isothermen bei verschiedenen c-Werte erhalten werden gleichzeitig verschiedene Modelle passen. Das richtige Modell ist neben basierend auf die Güte der Anpassung über die gesamte variable-c Datensatz identifiziert. Dieser Prozess ist hier, um die Aminoglykosid-Resistenz verursachenden Enzym Aminoglycosid N-6&#39;-Acetyltransferase-II (AAC (6 &#39;)-II) beantragt. Obwohl unsere Methode ist anwendbar auf jedem System, ist die Notwendigkeit, diese Strategie besser mit einem Makromolekül-Ligand-System zeigt Allosterie oder Kooperativität gezeigt, und wenn verschiedene verbindliche Modelle bieten im Wesentlichen identisch passt auf die gleichen Daten. Nach unserer Kenntnis gibt es keine derartigen Systeme im Handel erhältlich. AAC (6 &#39;)-II ist ein Homo-Dimer mit zwei aktiven Zentren, welche Kooperativität zwischen den beiden Untereinheiten. Allerdings ITC-Daten an einem einzigen c-Wert erhalten kann ebenso gut fit sein, um mindestens zwei verschiedene Modelle einer Zwei-sets-of-Sites eigenständiges Modell und eine Zwei-Seiten sequentielle (Genossenschaft) Modell. Durch Variation der c-Wert wie oben erläutert, wurde festgestellt, dass die korrekte Bindung Modell für AAC (6 &#39;)-II a two-site sequenzielle Bindung Modell ist. Hier beschreiben wir die Schritte, die bei der Durchführung von ITC Experimenten werden, um Datensätze für variable-c-Analysen erhalten müssen.

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 ist ein leistungsfähiges Werkzeug für die Untersuchung der Bindung eines Liganden an seinen Wirt. In komplexen Systemen kann jedoch mehrere Modelle die Daten gleich gut passen. Die hier beschriebene Methode bietet eine Möglichkeit, die entsprechende Bindung Modell für komplexe Systeme aufzuklären und extrahieren Sie die entsprechende thermodynamische Parameter.

Sammeln Variable-Konzentration Isotherme Titrationskalorimetrie Datasets in Auftrag, um bindende Mechanismen bestimmen

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 Aufrufe.  McGill University. ITC ist ein leistungsfähiges Werkzeug für die Untersuchung der Bindung eines Liganden an seinen Wirt. In komplexen Systemen kann jedoch mehrere Modelle die Daten gleich gut passen. Die hier beschriebene Methode bietet eine Möglichkeit, die entsprechende Bindung Modell für komplexe Systeme aufzuklären und extrahieren Sie die entsprechende thermodynamische Parameter.

Serie: 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.

Titration von Human Coronaviren Mit einem Immunoperoxidase Assay

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

Forschung

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.

Sammeln und Messen Nozizeptive und Entzündungsmediatoren in chirurgische Wunden

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.

Die Nutzung der Protease Fluorescent Detection Kit für Protease-Aktivität zu bestimmen

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 - Ein Verfahren zur Transkriptom-weiten die Bindungsstellen des RNA-bindende Proteine ​​Identifizieren

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.

Isotherme Titrationskalorimetrie für Mess-Makromolekül-Ligand-Affinität

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.

Kultivierung primären Ratten Inner Medulläre Sammelkanal Cells

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.

Biochemische Titration von Glykogen<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.

Fluoreszenzbasierten Primer Extension-Technik, um Transkriptionsstartpunkte und Schnittstellen von RNasen Bestimmen<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.

Messung und Analyse von extrazellulären Säureproduktion zu ermitteln glykolytischen Rate

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.