Name: automated hydrophobic interaction chromatography column selection for use in protein purification
Uploaded: 2011-09-21T13:00:00.000+00:00
Duration: 10 min 21 s
Description: Watch this Scientific Journal Video about Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification at JoVE.com

Diese Arbeit wurde vom National Institutes of Health Zuschuss GM086822 und National Science Foundation Großforschungseinrichtungen Instrumentation Grant DBI-0960313 gefördert. Die Autoren bedanken sich bei Dr. danken. Jon Miyake &amp; Donna Hardy (Bio-Rad) und Jennifer Loertscher (Seattle University) für ihre fachliche Unterstützung. Wählen Chromatographiereagenzien und ergänzende Instrumente wurden großzügigerweise von Bio-Rad zur Verfügung gestellt.

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	<li>Cummins, P. M., O'Connor, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophobic+interaction+chromatography.">Hydrophobic interaction chromatography.</a> Methods Mol. Biol. 681, 431-437 (2011).</li><li>Nagrath, D., Xia, F., Cramer, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+modeling+of+nonlinear+hydrophobic+interaction+chromatographic+systems.">Characterization and modeling of nonlinear hydrophobic interaction chromatographic systems.</a> J. Chromatogr. A. 1218, 1219-1226 (2011).</li><li>To, B. C., Lenhoff, 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=Hydrophobic+interaction+chromatography+of+proteins.+IV.+Protein+adsorption+capacity+and+transport+in+preparative+mode.">Hydrophobic interaction chromatography of proteins. IV. Protein adsorption capacity and transport in preparative mode.</a> J. Chromatogr. A. 1218, 427-440 (2011).</li><li>Huddleston, J. G., Wang, R., Lyddiatt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+use+of+mild+hydrophobic+interaction+chromatography+for+"method+scouting"+protein+purification+strategies+in+aqueous+two-phase+systems:+a+study+using+model+proteins.">On the use of mild hydrophobic interaction chromatography for "method scouting" protein purification strategies in aqueous two-phase systems: a study using model proteins.</a> Biotechnol Bioeng. 44, 626-635 (1994).</li><li>Arun, K. H., Kaul, C. L., Ramarao, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+proteins+in+receptor+research:+an+emerging+tool+for+drug+discovery.">Green fluorescent proteins in receptor research: an emerging tool for drug discovery.</a> J. Pharmacol. Toxicol. Methods. 51, 1-23 (2005).</li><li>Terrizzi, S. C., Banh, C., Brossay, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L.A+Protocol+for+the+Production+of+KLRG1+Tetramer.">L.A Protocol for the Production of KLRG1 Tetramer.</a> J. Vis. Exp. (35), e1701-e1701 (2010).</li><li>Murphy, P. J. M., Morishima, Y., Kovacs, J. J., Yao, T. P., Pratt, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+dynamics+of+hsp90+action+on+the+glucocorticoid+receptor+by+acetylation/deacetylation+of+the+chaperone.">Regulation of the dynamics of hsp90 action on the glucocorticoid receptor by acetylation/deacetylation of the chaperone.</a> J. Biol. Chem. 280, 33792-33799 (2005).</li><li>Zeytuni, N., Zarivach, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+the+M.+magneticum+strain+AMB-1+Magnetosome+Associated+Protein+MamA&#916;41.">Purification of the M. magneticum strain AMB-1 Magnetosome Associated Protein MamA&#916;41.</a> J. Vis. Exp. (37), 10-3791 (2010).</li><li>Kong, Y., Li, X., Bai, G., Ma, G., Su, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automatic+system+for+multidimensional+integrated+protein+chromatography.">An automatic system for multidimensional integrated protein chromatography.</a> J. Chromatogr. A. 1217, 6898-6904 (2010).</li><li>Camper, D. V., Viola, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+automated+protein+purification.">Fully automated protein purification.</a> Anal. Biochem. 393, 176-181 (2009).</li><li>Hammon, J., Palanivelu, D. V., Chen, J., Patel, C., Minor, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+green+fluorescent+protein+screen+for+identification+of+well-expressed+membrane+proteins+from+a+cohort+of+extremophilic+organisms.">A green fluorescent protein screen for identification of well-expressed membrane proteins from a cohort of extremophilic organisms.</a> Protein Sci. 18, 121-133 (2009).</li><li>Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+green+and+red+fluorescent+proteins+to+teach+protein+expression,+purification,+and+crystallization.">Using green and red fluorescent proteins to teach protein expression, purification, and crystallization.</a> Biochem. Mol. Biol. Educ. 36, 43-54 (2008).</li><li>Queiroz, J. A., Tomaz, C. T., Cabral, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophobic+interaction+chromatography+of+proteins.">Hydrophobic interaction chromatography of proteins.</a> J. Biotechnol. 87, 143-159 (2001).</li><li>McCue, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+and+use+of+hydrophobic+interaction+chromatography+in+protein+purification+applications.">Theory and use of hydrophobic interaction chromatography in protein purification applications.</a> Methods Enzymol. 463, 405-414 (2009).</li></ol>

Wählen Chromatographiereagenzien und ergänzende Instrumente wurden von Bio-Rad zur Verfügung gestellt.

Flüssigchromatographie Techniken wurden von unschätzbarem Wert zur Herstellung von hoch gereinigten Proteinen notwendig für die Durchführung von immunologischen 6, biochemische 7, 8 und strukturelle Studien nachgewiesen. HIC Reinigungsverfahren meist eine empirische Bestimmung einer bevorzugten Medium und Ligandenstruktur, Ligandendichte, und Matrix Wulst Eigenschaften alle haben gezeigt, dass chromatographischen Ergebnissen 2, 3 auswirken. Automatisierte Spalte Scouting ist ein effizienter Ansatz für die Auswahl eines HIC-Medium für die spätere Optimierung und Reinigung von Proteinen 4. Die automatisierte Spalte Scouting vorgestellte Methode kann leicht an verschiedene HIC Proteinreinigung Strategien angepasst werden. Änderungen der Salzkonzentration, die Wahl von Salz, Salzgradienten und pH-Wert kann weiter zu verbessern Reinigungsbedingungen, und die Wirkungen der Variation dieser wichtigen Parameter wurden bereits 13,14 Bewertung. HIC Eluats, das eine teilweise gereinigte Ziel proteIn kann weiter gereinigt werden unter Verwendung eines komplementären chromatographischen Technik, z. B. Ionenaustauschchromatographie (IEX) oder Gelfiltration / Größenausschlusschromatographie 9,10. Aufgrund seiner einzigartigen Licht-Absorption und Emission Eigenschaften kann das Elutionsprofil von GFP identifiziert mit In-Line-und Nachlauf Ansätze werden. Zu diesem Zweck kann das Protokoll weiter zur Reinigung von rekombinanten GFP-Fusionsproteine ​​angepasst werden, bei dem eine unabhängige Zielprotein &quot;Tag&quot; mit GFP. GFP-markierten Target-Proteine ​​können mit UV-Licht 11 und unter Verwendung verschiedener chromatographischer Methoden, einschließlich der HIC Reinigungsstrategie oben beschrieben detektiert werden. GFP Reinigung ist auch zu einem pädagogischen Grundnahrungsmittel in Biochemie-Labors für die Lehren der modernen Wissenschaft Techniken Protein 12. Während grundlegende HIC Proteinreinigung können unter Verwendung eines im wesentlichen weniger robust Chromatographie sy werdenStamm weist die Instrumentierung hier vorgestellten eine Reihe von Vorteilen zur Erlangung günstige Ergebnisse zu unterstützen. Bemerkenswerte Vorteile der Verwendung eines hoch automatisiertes System umfassen die Run-to-Run Zustand Reproduzierbarkeit, Zeitersparnis verbessert, und verringert Chancen für Luft in das System 10 eingeführt werden. System-gesteuerten Puffer Mischung, die durch das System Maximalstelle erleichtert wird, ermöglicht eine erhöhte Konsistenz in Puffer Herstellung und experimentelle Reproduzierbarkeit. Zeichnung genügend Probe in die dynamische Last Probenschleife für alle Scouting läuft weiter eine Übereinstimmung der Probe, die zu jeder Spalte hinzugefügt und ermöglicht es beim sequentiellen läuft ohne Unterbrechung oder automatisch geladen. Kontrollierte Probeninjektion aus der Probenschleife auf die Säule sinkt Variabilität, die bei manueller Beladung Probe auftreten kann. Ein Paar von Spaltenauswahlleitungen Ventile ermöglichen aufeinanderfolgenden Probe läuft, von denen jeder mit einem anderen HIC-Säule, ohne das System replumb. Darstellende mul<html> ti-Wellenlängen-Analyse ist besonders vorteilhaft, wenn das Testen einer Protein mit einer einzigartigen spektrophotometrische Profil. Zusätzlich zu GFP kann Cytochromen, Flavoproteinen und anderen Häm-Proteinen von dieser Technik profitieren. Leitfähigkeit und pH-Überwachungseinrichtungen können für die Überprüfung der Echtzeit experimentellen Bedingungen. Split Bruchteil Eluat Sammlung ermöglicht eine verbesserte Bruchteil Handhabung und ermöglicht die einfache Übertragung für Post-Run Analysemethoden, die eine minimale Probenvolumen (zB ELISA, SDS-PAGE, Western Blot, und Experion Mikrofluidik-Elektrophorese) erfordern. Beim Betrieb der zweiten Fraktionssammler offline erfordert manuelle Synchronisation, ermöglicht es maximale Flexibilität bei der Auswahl Fraktionssammler. Die bedeutendsten Nachteile bei Verwendung eines solchen robusten Chromatographie-System für die HIC-Säule Scouting und Reinigung von Proteinen gehören die Anfangszeit und die Haushaltsausgaben mit Instrument Erwerb und Bedienerschulung assoziiert. t &quot;&gt; Das hier vorgestellte Protokoll nutzt eine Bio-Rad DuoFlow Chromatographie-System;. aber ebenso robuste Messgeräte anderer Hersteller, wie zB die ÄKTA Avant von GE Healthcare, können ebenfalls verwendet werden und sind in der Lage gleichwertige Ergebnisse sogar vergleichbar Chromatographie-System haben einzigartige Eigenschaften (z. B. Methode der Programmierung Komponente Nomenklatur, der Präferenz des Betreibers und Skalierbarkeit Einschränkungen), die vor Einleitung einer Reinigungsprozedur oder Instrument Erwerb berücksichtigt werden sollten.

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> <a href="http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=223301" target="_blank">BioLogic DuoFlow Pathfinder 20 System</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> <a href="http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=223301" target="_blank">7602257</a> </td><td> Inklusive System Maximizer, Mixer, F10-Workstation, AVR7-3 Ventil, Quadtec UV / Vis-Detektor mit Durchflusszelle, BioFrac Fraktionssammler, BioLogic Software und Starter-Kit </td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=760-0408&vertical=LSR&parentCategoryGUID=22c495f1-36ff-44a3-9140-ee7e54d0b1ba" target="_blank">Avr9-8-Stream-Ventil wählen</a><html></td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=760-0408&vertical=LSR&parentCategoryGUID=22c495f1-36ff-44a3-9140-ee7e54d0b1ba" target="_blank">7600408</a> </td><td> Hochdruckventil, 9-Port, 8-Stellung, 3500 psi (233 bar) begrenzen, zur Verwendung mit biologischen System DuoFlow </td></tr><tr><td> Umschaltventil SV3T-2 </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> 7600410 </td><td> Magnetventil, 3-Port, 2-Stellung, 30 psi (2 bar) begrenzen, zur Verwendung mit biologischen System DuoFlow </td></tr><tr><td> Stream-Verteilerventil </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td></td><td></td></tr><tr><td>Tical LSR = &amp; = parentCategoryGUID 22c495f1-36ff-44a3-9140-ee7e54d0b1ba &quot;target =&quot; _blank &quot;&gt; dynaloop 25 Kit </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=750-0451&vertical=LSR&parentCategoryGUID=22c495f1-36ff-44a3-9140-ee7e54d0b1ba" target="_blank">7500451</a> </td><td> Sliding-Kolben-Probenschleife Kit enthält 25 ml dynaloop Gleitkolben Schleife, dynaloop Teilesatz (# 750-0450) </td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/LSR/PDP/ec82e7b4-919e-428b-ac03-9adf1f50a48e/BioFractrade_Fraction_Collector" target="_blank">BioFrac Fraktionssammler</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/LSR/PDP/ec82e7b4-919e-428b-ac03-9adf1f50a48e/BioFractrade_Fraction_Collector" target="_blank">7410002</a> </td><td> Zwei Bruchteil col<html> Lektoren eingesetzt </td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=741-0088&vertical=LSR&parentCategoryGUID=ec82e7b4-919e-428b-ac03-9adf1f50a48e" target="_blank">BioFrac Mikroplatten-Drop-Kopf-Adapter</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=741-0088&vertical=LSR&parentCategoryGUID=ec82e7b4-919e-428b-ac03-9adf1f50a48e" target="_blank">7410088</a> </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&vertical=LSR&country=US&lang=en&productID=741-0017" target="_blank">BioFrac Mikrotiterplatte Adapter</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td>&amp; Country = US &amp; lang = de &amp; ProductID = 741-0017 &quot;target =&quot; _blank &quot;&gt; 7410017 </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=750-0202&vertical=LSR&parentCategoryGUID=c140016b-1958-4a91-be4c-8645a4c8204f" target="_blank">UV-Optik-Modul</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=750-0202&vertical=LSR&parentCategoryGUID=c140016b-1958-4a91-be4c-8645a4c8204f" target="_blank">7500202</a> </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=760-1331&vertical=LSR&parentCategoryGUID=c4095dc1-9260-4431-b066-823b050048c8" target="_blank">Halogenlampe</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> &lt;<html> ein target = &quot;_blank&quot;&gt; 7601331 </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=220901" target="_blank">Econo Gradientenpumpe</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=220901" target="_blank">7319001</a> </td><td> Gradientenpumpe für Niederdruck-Reinigung von Proteinen, enthält Schläuche und Armaturen-Kit </td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=224301" target="_blank">Experion-System</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td>bio-rad.com/prd/en/US/adirect/biorad? cmd = BRCatgProductDetail &amp; ProductID = 224301 &quot;target =&quot; _blank &quot;&gt; 7007001 </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=224101" target="_blank">Experion Pro260 Analysis Kit</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=224101" target="_blank">7007102</a> </td><td></td></tr><tr><td> HiTrap HIC-Säule Auswahl-Kit </td><td> GE Healthcare </td><td> 28-4110-07 </td><td> Inklusive 7 x 1 ml vorgepackte Säulen des HIC Medien für Scouting </td></tr><tr><td> GE Healthcare-to-Bio-Rad Spalte Armaturen </td><td> GE Healthcare </td><td> 18111251 und 18111257 </td><td></td></tr></tbody></table>

automated hydrophobic interaction chromatography column selection for use in protein purification

Im Gegensatz zu anderen chromatographischen Methoden zur Aufreinigung von Proteinen (zB Gelfiltration, Affinität und Ionenaustausch), hydrophobe Interaktions-Chromatographie (HIC) erfordert häufig experimentelle Bestimmung (so genannte Screening oder &quot;Scouting&quot;), um die am besten geeigneten chromatographischen Medium wählen zur Reinigung eines bestimmten Proteins 1. Die hier vorgestellte Methode beschreibt ein automatisiertes Konzept für Scouting für eine optimale HIC-Medien in Proteinreinigung verwendet werden. HIC trennt Proteine ​​und andere Biomoleküle aus einer rohen Lysat auf Unterschiede in der Hydrophobie der Basis. Ähnlich wie Affinitätschromatographie (AC) und Ionenaustauschchromatographie (IEX), HIC kann die Konzentration des Proteins von Interesse, da sie durch das chromatographische voranschreitet. Proteine ​​besten für die Reinigung durch HIC geeignet sind, umfassen solche mit hydrophoben Oberflächenbereiche und in der Lage, um die Exposition zu widerstehen, um Konzentrationen von mehr als 2 M Salz Munitionnium-Sulfat ((NH 4) 2 SO 4). HIC wird oft als ein Reinigungsverfahren für Proteine ​​fehlt ein Affinitäts-Tag gewählt und somit ungeeignet für Wechselstrom, und wenn IEX keine ausreichende Reinigung bereitzustellen. Hydrophobe Reste, die an das Protein binden Deckschicht zeitweilig in eine unpolare Liganden, der mit einem inerten, unbeweglich Matrix. Die Wechselwirkung zwischen Protein und dem Liganden sind stark abhängig von der Salzkonzentration des Puffers fließt durch die Chromatographiesäule, mit hohen Ionenkonzentrationen Stärkung der Protein-Ligand-Interaktion und dass das Protein unbeweglich (dh innerhalb der Säule gebunden) 2. Wie Salzkonzentrationen Abnahme, das Protein-Ligand-Wechselwirkung abführt, das Protein wieder wird mobil und von der Säule eluiert. Mehrere HIC Medien sind kommerziell erhältlich in vorverpackten Spalten, die jeweils eine von mehreren hydrophoben Liganden (zB S-Butyl, Butyl, Octyl und Phenyl) bei unterschiedlichen Dichten eine Agarose-Kügelchen mit einem spec vernetztIFIC Durchmesser 3. Automatisierte Spalte Scouting ermöglicht eine effiziente Methode zur Bestimmung des HIC-Medien für die Zukunft, mehr erschöpfend Optimierung Experimente verwendet werden sollte und Proteinreinigung läuft 4. Die spezifische Protein, das hier gereinigte rekombinante grün fluoreszierendes Protein (GFP), doch, kann dieser Ansatz zur Reinigung von anderen Proteinen, mit einem oder mehreren hydrophoben Oberflächenbereiche ausgebildet werden. GFP dient als nützliches Modell Protein aufgrund seiner Stabilität, einzigartige Licht Absorptionspeak bei 397 nm und Fluoreszenz, wenn sie mit UV-Licht 5 ausgesetzt. Bakterienlysates Wildtyp-GFP wurde in einem Hochsalz-Puffer hergestellt, geladen in einem Bio-Rad DuoFlow Mitteldruck-Flüssigkeits-Chromatographie-System und adsorbiert HiTrap HIC-Säulen, die unterschiedliche HIC Medien. Das Protein wurde von den Säulen eluiert und mittels in-line und Nachlauf Nachweisverfahren. Buffer Blending, dynamisches Probenschleife Injektion, sequentielle colUMN Auswahl, Multi-Wellenlängen-Analyse, und getrennte Fraktion angesehen Eluat erhöht die Funktionalität des Systems und Reproduzierbarkeit der experimentellen Ansatz.

Watch this Scientific Journal Video about Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification at JoVE.com

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

Ein automatisiertes Verfahren zur Identifizierung von geeigneten hydrophoben Interaktionschromatographie (HIC) Medien, die in dem Verfahren der Proteinreinigung verwendet werden vorgestellt. Das Verfahren nutzt eine Mitteldruck-Flüssigkeits-Chromatographie-System einschließlich automatisierter Puffer Blending, dynamisches Probenschleife Injektion, sequentielle Spalte Auswahl, Multi-Wellenlängen-Analyse-und Split-Fraktion Eluat Sammlung.

Automatisierte Hydrophobe Interaktions-Chromatographie Spaltenauswahl für den Einsatz in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

automated-hydrophobic-interaction-chromatography-column-selection-for

Research

JoVE Journal

Biology

43.5K Aufrufe.  Seattle University. Ein automatisiertes Verfahren zur Identifizierung von geeigneten hydrophoben Interaktionschromatographie (HIC) Medien, die in dem Verfahren der Proteinreinigung verwendet werden vorgestellt. Das Verfahren nutzt eine Mitteldruck-Flüssigkeits-Chromatographie-System einschließlich automatisierter Puffer Blending, dynamisches Probenschleife Injektion, sequentielle Spalte Auswahl, Multi-Wellenlängen-Analyse-und Split-Fraktion Eluat Sammlung.

Serie: Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

Expression, Reinigung, Kristallisation und Enzym-Assays von Fumarylacetoacetat-Hydrolase-Domänen-haltigen Proteinen

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Forschung

Biochemie

An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is also a key component in the development of new therapeutics. The evolving era of functional proteomics is fueling the demand for high-throughput protein purification and improved techniques to facilitate this. It was hypothesized that a multi column plate adaptor (MCPA) can interface multiple chromatography columns of different resins with multi-well plates for parallel purification. This method offers an economical and versatile method of protein purification that can be used under gravity or vacuum, rivaling the speed of an automated system. The MCPA can be used to recover milligram yields of protein by an affordable and time efficient method for subsequent characterization and analysis. The MCPA has been used for high-throughput affinity purification of SH3 domains. Ion exchange has also been demonstrated via the MCPA to purify protein post Ni-NTA affinity chromatography, indicating how this system can be adapted to other purification types. Due to its setup with multiple columns, individual customization of parameters can be made in the same purification, unachievable by the current plate-based methods.

A multi-column plate adapter allows chromatography columns to be interfaced with multi-well collection plates for parallel affinity or ion exchange purification providing an economical high throughput protein purification method. It can be used under gravity or vacuum yielding milligram quantities of protein via affordable instrumentation.

Ein wirtschaftliches und vielseitiges Hochdurchsatz-Proteinreinigungssystem mit einem Mehrsäulenplattenadapter

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is ...

Chromatography-based Biomolecule Purification Methods

In biochemistry, chromatography-based purification methods are employed to isolate compounds from a complex mixture. Two such methods used commonly by biochemists are size-exclusion chromatography and affinity chromatography. In size-exclusion chromatography, a column packed with porous beads separates components of a mixture based on size. On the other hand, affinity chromatography allows for a more specific separation of biomolecules by using a column that is composed of stationary phase, which contains target-specific ligands.
This video serves as an introduction to size-exclusion and affinity chromatography, as well as the concepts that govern them. A step-by-step procedure for the purification of a histidine-tagged protein by immobilized metal affinity chromatography is described. Applications for both of these chromatographic methods in biochemistry and biomedical research are also profiled.
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&#34;Chromatography&#34; refers to a wide range of methods used to isolate a component from a complex mixture, an essential step before a biomolecule&#39;s properties and activities can be determined. Each chromatographic technique has a different mechanism for separation, depending on the sample matrix and target compound. This video will focus on the principles and operation of two methods common to biochemistry: size-exclusion and affinity chromatography.
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<div class="chapter_text" id="script_1">
Size-exclusion chromatography or SEC is based on the size of the compounds in the sample. A mobile phase containing the sample is added to a column with a porous material: the stationary phase. The molecules in the sample fall into 1 of 3 categories.
Molecules too large to enter the pores travel the shortest distance through the column. Any species with a molecular weight above this &#34;exclusion limit&#34; will exit the column at the same time. Molecules small enough to freely enter the pores will be retained the longest, and will exit the column together. The molecular weight allowing complete pore entry is the &#34;permeation limit&#34;.
Only molecules between these limits will be separated from one another, as they spend varying amounts of time diffusing into and out of the pores. Smaller molecules are retained longer on the column because they spend more time in the stationary phase, whereas larger molecules within these limits exit earlier.
Molecular weights across 1 to 2 orders of magnitude fall within these limits. Columns are chosen with this in mind, or multiple columns can be used in series if there is a wide range of desired compounds.
Now that you&#39;ve seen the theory of SEC, let&#39;s look at how it is carried out.
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<div class="chapter_text" id="script_2">
To begin the SEC procedure, the column must be equilibrated with deionized water and chromatography buffer. Once prepared, buffer containing the sample is injected onto the column. The buffer is then pushed through at a low flow rate. A detector monitors what exits the column to determine the presence of the desired analyte. Large molecules with molecular weights above the exclusion limit exit the column at the same time. Small fractions from the column are collected in tubes. Each fraction is tested for the quality of the target molecule by gel electrophoresis or other analytical techniques.
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<div class="chapter_text" id="script_3">
Now let&#39;s have a look at affinity chromatography or AC, one of the most efficient ways to purify proteins. Many biomolecules bind selectively to certain ligands-a property which AC utilizes by adhering a target-specific ligand to the stationary phase.
When the mixture flows through the column, the target molecules attach to the ligand, and the rest flow through. After the mixture has passed through the column, the target molecule can be collected through one of two elution methods based on specificity.
Biospecific elution can be said to a have a &#34;normal-&#34; or &#34;reverse-role&#34;. In normal-role biospecific elution, an agent is added that competes with the adhered ligand to bind with the target biomolecule.
In reverse-role biospecific elution, an agent competes with the target to bind to the adhered ligand. The second elution type, nonspecific elution, lowers the target-to-ligand binding by changing the solution&#39;s pH, ionic strength, or polarity. If a protein does not bind to a ligand that can be immobilized, the protein can be expressed containing a &#34;tag&#34;: short peptide sequences engineered to bind to the ligand.
One variety is immobilized metal ion affinity chromatography, &#34;IMAC&#34; for short, where an adhered metal ligand, like nickel or cobalt, binds to histidine residues on the modified protein. Through molecular biology techniques, target proteins are generated with repeating histidine residues, called a polyhistidine-tag, which binds to the metal via the imidazole side chain on histidine. Once bound, the protein can be collected with free imidazole via reverse-role specific elution and later used in a wide variety of downstream applications.&#160;Now that you&#39;ve seen the theory of affinity chromatography, let&#39;s look at an IMAC procedure in the laboratory.

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<div class="chapter_text" id="script_4">
In IMAC, the stationary phase can be added directly to the mixture of mobile phase and sample, allowing the binding of the his-tagged protein. This slurry is then poured into the column, where the non-bound compounds drip into waste, while the slurry remains. The slurry container is rinsed to collect residual resin and sample, which is added to the column.
The resin is stirred to ensure the unbound components are free-flowing. Added wash buffer helps flush them away. Once all of the unbound components have been removed, the waste is replaced with a container to collect the target protein.
Buffer containing imidazole is added, stirred, and allowed to rest to unbind the target molecule. The imidazole binds to the metal, replacing and releasing the tagged protein. The freed protein is collected, and the imidazole step is repeated to ensure total collection. To further purify the sample, SEC can be run on the sample prior to analysis.
</div>
<div class="chapter_text" id="script_5">
Now that we&#39;ve seen the theory and procedure of these two techniques, let&#39;s look at some of the ways they&#39;re applied in the biochemical field.
A common reason to purify proteins is to study their role in disease. Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator protein, or CFTR. After growing the protein with a his-tag in yeast, both affinity and size-exclusion chromatography allow the isolation of the protein, followed by the study of its function.
In some instances, the presence of a polyhistidine tag can change a protein&#39;s structure, thereby affecting its function. Another common tag is maltose-binding protein or MBP, which will bind to bound amylose in a column. Maltose is then used to release the complex. The MBP can then be cleaved and removed with SEC to produce the pure desired protein.
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<div class="chapter_text" id="script_6">
You&#39;ve just watched JoVE&#39;s video on size-exclusion and affinity chromatography. It covered the theory of the techniques, went over general procedures, and covered some of the uses of the techniques.
Thanks for watching!
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Size Exclusion and Affinity Chromatography

Chromatographie-basierte Biomolekül Reinigungsverfahren

In biochemistry, chromatography-based purification methods are employed to isolate compounds from a complex mixture. Two such methods used commonly by ...

Lehre

JoVE Science Education

Chemie

Grundlagen der Biochemie

Nickel Affinity Chromatography-Based Protein Purification: A Technique to Purify Polyhistidine-Tagged Recombinant Proteins from Bacterial Cell Lysate

Source: Pokhrel, A. et al. A <a href="https://www.jove.com/v/58547">Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile</a>. J. Vis. Exp. (2018)
In this video, we demonstrate the nickel affinity chromatography technique to purify histidine-tagged pyrophosphokinase enzymes from Clostridium difficile bacteria.

Proteinreinigung auf Basis der Nickel-Affinitätschromatographie: Eine Technik zur Aufreinigung von Polyhistidin-markierten rekombinanten Proteinen aus bakteriellem Zelllysat

Source: Pokhrel, A. et al. A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile. J. Vis. Exp. (2018)
In this ...

JoVE Encyclopedia of Experiments

Biological Techniques

Chromatography Techniques

Butyl-Dependant Hydrophobic Interaction Liquid Chromatography: A Technique to Characterize Antibody-Drug Conjugates Based on Hydrophobic Interactions

Source: Oller-Salvia, B. <a href="https://www.jove.com/v/58066">Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction</a>. J. Vis. Exp. (2018).
This video demonstrates the hydrophobic interaction liquid chromatography-based characterization of antibody-drug conjugates. Using a butyl ligand-based chromatography column, the antibody-drug conjugates are characterized via hydrophobic interactions with the ligand.

Butyl-abhängige hydrophobe Wechselwirkung Flüssigkeitschromatographie: Eine Technik zur Charakterisierung von Antikörper-Wirkstoff-Konjugaten auf der Grundlage hydrophober Wechselwirkungen

Source: Oller-Salvia, B. Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal ...

An Affinity Chromatography Technique for the Purification of Monoclonal Antibodies

Source: Elgundi, Z., et al. <a href="https://app.jove.com/v/55153">Laboratory Scale Production and Purification of a Therapeutic Antibody.</a> J. Vis. Exp. (2017).
This video demonstrates a technique for purifying monoclonal antibodies from the culture supernatant obtained from an antibody-producing cell culture. The process involves using a matrix of crosslinked agarose beads with conjugated Protein A, which immobilizes the antibodies in the supernatant on the beads, allowing for effective separation from contaminants. By applying an elution buffer with low pH, the protein A-antibody interaction is loosened, facilitating the collection of antibody-containing fractions.

Eine Affinitätschromatographie-Technik zur Aufreinigung von monoklonalen Antikörpern

1. Antibody Purification by Affinity Chromatography Using an Automated Fast Protein Liquid Chromatography (FPLC) System
NOTE: The following procedure can ...

Immunology

Antibody-Based Technologies

Production and Purification

Purification of Self-Assembling Protein Nanoparticles using Affinity Chromatography

Source: Karch, C. P. et al., <a href="https://app.jove.com/v/60103">Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation.</a>&#160;J. Vis. Exp. (2019)
This video showcases the purification of histidine-tagged self-assembled protein nanoparticles or SAPNs using affinity liquid chromatography. The resulting purified SAPN fractions are suitable for vaccine development, holding promise for potential immunotherapeutic applications.

Aufreinigung von selbstorganisierenden Protein-Nanopartikeln mittels Affinitätschromatographie

1. Expression of the Self-assembling protein nanoparticles core containing a six-helix bundle (SHB-SAPN) Protein in E. coli BL21(DE3)

	Mix 95 mL of ...

Immunotherapy

Immunogenics and Vaccine Development

Types Of Column Chromatography

The stability and compatibility of column material with samples are crucial for efficient purification in chromatographic techniques. Various operating parameters such as pH, temperature, or solvent affect the packing of the column material, thereby determining the purification efficiency. The choice of column material also plays an essential role in deciding the operating parameters and can be modified based on the proteins that need to be purified.
Gel Filtration Chromatography 
When the protein&#39;s chemical nature is unknown, gel filtration chromatography is used for purification based on size. Matrices such as agarose, polyacrylamide, dextran derivatives, or silica beads are used for low-pressure systems, whereas polymeric resins are used for higher flow rates. The varying degree of cross-linking of these polymeric materials determines the pore size of the matrix, which decides the size range of proteins that can be separated.
Ion Exchange Chromatography
Ion-exchange chromatography can purify proteins based on their net charge at a particular pH determined by their isoelectric point or pI. The net charge on matrix resin is used to purify oppositely charged proteins by replacing the cations or anions from the matrix resin with the charged proteins. The resins are categorized based on the type of ion that is exchanged &#8212; positively charged proteins replace cations from cation-exchange resins, and vice versa. Strong ion exchangers with stability over a wide range of buffer pH include quaternary ammonium, sulfonate, and sulfopropyl resins. In contrast, weak ion exchangers that are effective only in a narrow pH range include diethyl aminoethyl (DEAE) or carboxymethyl (CM) resins.
Affinity Chromatography
Affinity chromatography involves immobilizing protein-specific antibodies or enzyme-specific substrates to the column material, usually agarose beads. For example, a column with the immobilized antibody of choice is prepared to separate specific antigens. When the mixed antigen sample is passed through the&#160; column, the target antigens bind to the immobilized antibodies and separate from the mixture. To separate recombinant proteins tagged with enzymes such as beta-galactosidase, the corresponding substrate Isopropyl-&#946;-D-thiogalactopyranoside (IPTG) is immobilized on the column to purify the enzyme.
High-pressure liquid chromatography (HPLC) and gas chromatography (GC) are the most commonly used column chromatography techniques. In HPLC, the solvent flows at high pressure through a tightly packed column that improves efficiency. Gas chromatography analyzes volatile compounds by converting them to their gaseous forms upon heating. The volatilized sample is pushed through the column by inert gasses such as nitrogen and helium.

Arten der Säulenchromatographie

The stability and compatibility of column material with samples are crucial for efficient purification in chromatographic techniques. Various operating ...

JoVE Core

Zellbiologie

Unit 1

Analysieren von Zellen und Proteinen

SCHLÜSSELBEGRIFFE UND KONZEPTE

Anion-Exchange Chromatography-Based Protein Purification: A Separation Technique to Isolate a Protein of Interest From Dialyzed Bacterial Lysate Based on Net Charge

Start the FPLC with general maintenance. Connect column buffers AIEX-A and AIEX-B to buffer valves, and the anion-exchange resin-containing column to the column ports of the FPLC. Adjust the general chromatographic parameters accordingly.
Start the AIEX program in the FPLC software and choose the equilibration volume for buffer A. Subsequently, load the sample onto the column, and elute the proteins with a linear gradient from 0% to 100% high-salt buffer. Collect 2-milliliter fractions during elution.
Analyze the eluted fractions after finishing the run.

Proteinreinigung auf Basis der Anionenaustauschchromatographie: Eine Trenntechnik zur Isolierung eines Proteins von Interesse aus dialysiertem bakteriellem Lysat auf der Grundlage der Nettoladung

EoE Sub Articles

Calcium-Dependent Hydrophobic Interaction Chromatography: A Technique to Purify Calcium-Binding Proteins Based on Hydrophobic Interactions

To continue the protein purification, after the protein dialysis, add calcium chloride to the sample to a final concentration of 25 millimolar, which will facilitate binding of S100A12 to the resin in the next step. Then, filter the sample through a filter with a pour size of 0.45 microns.
Equilibrate HIC buffers and samples to 4 degrees Celsius. Next, connect column buffers HIC-A and B and the column, to the system and adjust the parameters.
Start the method to equilibrate the column with 1 to 2 column volumes of buffer HIC-A, and load the sample, and the &#39;wash unbound sample block&#39; when the UV signal reaches baseline level again.
Then, start elution with a calcium chelator-containing buffer EDTA. Collect 2 milliliters of peak fractions during elution.

Calciumabhängige hydrophobe Interaktionschromatographie: Eine Technik zur Aufreinigung von Calcium-bindenden Proteinen auf der Grundlage hydrophober Wechselwirkungen

Automatisierte Hydrophobe Interaktions-Chromatographie Spaltenauswahl für den Einsatz in Protein Purification

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