Ce travail a été financé par les Instituts nationaux de la santé et de subvention GM086822 National Science Foundation majeure des instruments de recherche de subvention DBI-0960313. Les auteurs tiennent à remercier les Drs. Jon Miyake &amp; Donna Hardy (Bio-Rad) et Jennifer Loertscher (Seattle University) pour leur expertise technique. Sélectionnez réactifs de chromatographie et de l&#39;instrumentation supplémentaire a été généreusement fourni par la société Bio-Rad.

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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>

Sélectionnez réactifs de chromatographie et de l&#39;instrumentation supplémentaire ont été offerts par Bio-Rad.

Les techniques de chromatographie liquide se sont avérés inestimables pour la préparation de protéines hautement purifiées nécessaires à la conduite 6 immunologiques, biochimiques 7, 8 et structurelles des études. Méthodes de purification HIC nécessitent le plus souvent la détermination empirique d&#39;un milieu privilégié, et la structure du ligand, la densité de ligand, et de la matrice des propriétés de perles ont toutes été montré à influer sur les résultats chromatographiques 2, 3. Colonne automatisé le scoutisme est une approche efficace pour la sélection d&#39;un support pour l&#39;optimisation ultérieure HIC et 4 de purification des protéines. La méthode de la colonne automatisé dépistage présenté peut être facilement adaptée à différents HIC stratégies de purification des protéines. Les modifications de la concentration en sel, le choix du sel, gradient de sel, et le pH peut en outre améliorer les conditions de purification, et les effets de la variation de ces paramètres essentiels ont déjà été examinés 13,14. Éluat contenant une HIC prote cible partiellement purifiéen peut encore être purifié en utilisant une technique de chromatographie en phase complémentaire, tels que chromatographie échangeuse d&#39;ions (IEX) ou filtration sur gel / chromatographie d&#39;exclusion de taille 9,10. En raison de son absorption de la lumière unique et des caractéristiques d&#39;émission, le profil d&#39;élution de la GFP peut être identifié à l&#39;aide en ligne et de post-terme des approches. À cette fin, ce protocole peut en outre être adaptée pour la purification de recombinants GFP-protéines de fusion, dans laquelle une protéine cible est sans rapport avec &quot;tag&quot; avec la GFP. Protéines cibles GFP-étiquette peut être détectée avec une lumière UV et 11 purifiée en utilisant diverses méthodes chromatographiques, y compris la stratégie de purification HIC décrit ci-dessus. Purification GFP est également devenu un aliment de base dans les laboratoires de biochimie pédagogique pour l&#39;enseignement des techniques modernes de la science des protéines 12. Alors que de base de purification des protéines HIC peut être effectuée en utilisant une chromatographie sensiblement moins robuste sytige, l&#39;instrumentation présentée ici possède un certain nombre d&#39;avantages distincts pour aider à l&#39;obtention de résultats favorables. Avantages notables de l&#39;utilisation d&#39;un système hautement automatisé comprennent l&#39;amélioration de fonctionner à fonctionner condition de reproductibilité, de gain de temps, et une diminution des possibilités de l&#39;air d&#39;être introduites dans le système 10. Buffer blending système contrôlé, qui est facilitée par le système Maximizer, permet une plus grande cohérence dans la préparation du tampon et de la reproductibilité expérimentale. Dessin de charge d&#39;échantillon suffisante dans la boucle d&#39;échantillonnage dynamique pour tous dépistage exécute en outre assure l&#39;uniformité de l&#39;échantillon étant ajouté à chaque colonne et permet pistes séquentielles sans interruption ou manuel de rechargement. Injection de l&#39;échantillon contrôlé à partir de la boucle d&#39;échantillonnage sur la colonne diminue la variabilité qui peut se produire avec un chargement manuelle de l&#39;échantillon. Une paire de soupapes de sélection de colonne pour permettre pistes consécutives, chacune échantillon en utilisant une colonne différente HIC, sans avoir à replumb le système. Exécution mul<html> ti-longueur d&#39;onde d&#39;analyse est particulièrement bénéfique lors du dosage d&#39;une protéine avec un profil unique spectrophotométrique. En plus de la GFP, les cytochromes, flavoprotéines et autres hème contenant des protéines peuvent bénéficier de cette technique. Dispositifs de surveillance de la conductivité et le pH permettent de vérifier en temps réel des conditions expérimentales. Collecte de l&#39;éluat de Split fraction permet une manipulation fraction améliorée et permet le transfert facile pour les méthodes d&#39;analyse post-terme qui nécessitent un volume d&#39;échantillon minime (par exemple ELISA, SDS-PAGE, western blot, et Experion microfluidique électrophorèse). Pendant le fonctionnement de la seconde fraction déconnecté collecteur nécessite une synchronisation manuelle, il permet une flexibilité maximale dans le choix collecteur de fractions. Les inconvénients les plus importants à l&#39;utilisation d&#39;un tel système de chromatographie robuste pour la colonne HIC le scoutisme et la purification des protéines incluent le temps initial et les dépenses budgétaires liées à l&#39;acquisition instrument et la formation des opérateurs. t &quot;&gt; Le protocole présenté ici utilise un Bio-Rad DuoFlow chromatographie système;. Cependant, l&#39;instrumentation aussi robuste d&#39;autres fabricants, tels que l&#39;Avant ÄKTA de GE Healthcare, peuvent également être utilisés et sont capables de produire des résultats équivalents système de chromatographie Même comparables ont des caractéristiques uniques de programmation (par exemple, la méthode, les limites nomenclature des composants, préférences de l&#39;opérateur, et l&#39;évolutivité) qui devraient être considérés avant d&#39;entamer une procédure de purification ou de l&#39;acquisition instrument.

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td></tr><tr><td> <a href="http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=223301" target="_blank">DuoFlow BioLogic Pathfinder 20 Système</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> Comprend un système de Maximizer, mélangeur, poste de travail F10, AVR7-3 de soupape, UV Quadtec / détecteur de Vis avec cellule d&#39;écoulement, collecteur de fractions BIOFRAC, logiciels BioLogic, et kit de démarrage </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-flux Sélectionnez la vanne</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> Haut-pression à la valve, 9-port, en position 8, 3500 psi (233 bars) limite, pour une utilisation avec un système DuoFlow BioLogic </td></tr><tr><td> Vanne de dérivation SV3T-2 </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> 7600410 </td><td> Electrovanne, 3-port, en position 2, 30 psi (2 bars) limite, pour une utilisation avec un système DuoFlow BioLogic </td></tr><tr><td> Volet clapet de répartition </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td></td><td></td></tr><tr><td>tique = 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> Glisse-piston kit boucle d&#39;échantillonnage, comprend 25 ml DynaLoop coulissant boucle piston, kit de pièces DynaLoop (# 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">Collecteur de fraction BIOFRAC</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> Deux col fraction<html> lecteurs utilisés </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 microplaque adaptateur de tête goutte</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">Adaptateur BIOFRAC plaque de microtitration</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td>&amp; Country = Etats-Unis &amp; lang = fr &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">Optique UV Module</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">Lampe halogène</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> &lt;<html> un 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 dégradé la pompe</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> une pompe à gradient pour la purification de protéines à basse pression, et un kit de tube comprend raccords </td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=224301" target="_blank">Experion système</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">Kit Experion Analyse Pro260</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 kit HIC sélection de la colonne </td><td> GE Healthcare </td><td> 28-4110-07 </td><td> Comprend 7 x 1 ml pré-emballés colonnes des médias HIC pour le scoutisme </td></tr><tr><td> Raccords de GE Healthcare colonne à Bio-Rad </td><td> GE Healthcare </td><td> 18111251 18111257 et </td><td></td></tr></tbody></table>

automated hydrophobic interaction chromatography column selection for use in protein purification

Contrairement à d&#39;autres méthodes chromatographiques pour les protéines purification (filtration sur gel, par exemple, l&#39;affinité et l&#39;échange d&#39;ions), chromatographie d&#39;interaction hydrophobe (HIC) nécessite souvent la détermination expérimentale (dénommé dépistage ou &quot;scouting&quot;) afin de sélectionner le support le plus approprié par chromatographie en phase pour purifier une protéine donnée 1. La méthode présentée ici décrit une approche automatisée pour le scoutisme pour un média optimales HIC à être utilisés dans la purification des protéines. HIC sépare les protéines et autres biomolécules à partir d&#39;un lysat brut basée sur les différences de caractère hydrophobe. Semblable à la chromatographie d&#39;affinité (AC) et chromatographie échangeuse d&#39;ions (IEX), CIH est capable de concentrer la protéine d&#39;intérêt à mesure qu&#39;elle progresse dans le processus chromatographique. Protéines qui conviennent le mieux pour la purification par HIC comprennent ceux qui sont avec les régions de surface hydrophobes et capables de résister à l&#39;exposition au sel concentrations supérieures à 2 M munitionssulfate de titane ((NH 4) 2 SO 4). HIC est souvent choisi comme un procédé de purification des protéines qui n&#39;ont pas un marqueur d&#39;affinité, et donc impropres à AC, et quand IEX ne parvient pas à fournir de purification adéquate. Fractions hydrophobes sur la surface de la protéine temporairement se lier à un ligand non polaire couplé à une inerte, matrice immobile. L&#39;interaction entre la protéine et le ligand sont fortement tributaires de la concentration en sel de la mémoire tampon qui coule à travers la colonne de chromatographie, avec de fortes concentrations ioniques renforcement de l&#39;interaction protéine-ligand et de faire la protéine immobile (c.-à-lié à l&#39;intérieur de la colonne) 2. Sous forme de sel concentrations diminution, l&#39;interaction protéine-ligand se dissipe, la protéine est de nouveau mobile et est éluée de la colonne. Plusieurs médias HIC sont disponibles dans le commerce pré-emballés colonnes, chacune contenant plusieurs ligands hydrophobes (par exemple S-butyle, butyle, octyle, et phényl) réticulé à différentes densités de billes d&#39;agarose d&#39;un specdiamètre ific 3. Colonne automatisé dépistage permet une approche efficace pour déterminer quels médias HIC devrait être utilisée pour de futures expériences d&#39;optimisation, les plus exhaustives et purification des protéines fonctionne 4. La protéine spécifique se purifier ici est une protéine recombinante fluorescente verte (GFP), mais l&#39;approche peut être adaptée pour la purification d&#39;autres protéines avec une ou plusieurs régions de surface hydrophobes. GFP sert une protéine modèle utile, en raison de sa stabilité, unique pic d&#39;absorbance de lumière à 397 nm et la fluorescence lorsqu&#39;ils sont exposés à la lumière UV 5. Lysat bactérien contenant la GFP de type sauvage a été préparé dans un tampon riche en sel, chargé dans un Bio-Rad moyennes DuoFlow système de pression chromatographie en phase liquide, et adsorbés sur des colonnes HiTrap HIC contenant différents médias HIC. La protéine a été élue des colonnes et analysées par en ligne et les méthodes de détection post-terme. Mélange tampon, dynamique boucle d&#39;injection de l&#39;échantillon, col séquentiellelonne de sélection, multi-longueur d&#39;onde d&#39;analyse et de collecte de l&#39;éluat scission fraction augmenté la fonctionnalité du système et la reproductibilité de la démarche expérimentale.

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

Procédé automatisé pour identifier appropriés chromatographie d&#39;interaction hydrophobe (HIC) médias à être utilisés dans le procédé de purification de protéine est présenté. La méthode utilise un système de moyenne pression chromatographie en phase liquide, y compris buffer blending automatisé, dynamique boucle d&#39;injection de l&#39;échantillon, sélection de la colonne séquentielle, multi-longueur d&#39;onde d&#39;analyse et de collecte de l&#39;éluat scission fraction.

Automatisé d&#39;interaction hydrophobe Chromatographie sur colonne de sélection pour l&#39;utilisation dans la purification des protéines

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

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43.1K vues.  Seattle University. Procédé automatisé pour identifier appropriés chromatographie d'interaction hydrophobe (HIC) médias à être utilisés dans le procédé de purification de protéine est présenté. La méthode utilise un système de moyenne pression chromatographie en phase liquide, y compris buffer blending automatisé, dynamique boucle d'injection de l'échantillon, sélection de la colonne séquentielle, multi-longueur d'onde d'analyse et de collecte de l'...

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

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Coloration des protéines dans les gels de Coomassie G-250, sans solvant et l&#39;acide acétique organique

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

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Biologie

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification de la M. magneticum Strain AMB-1 Protein magnétosome Associated MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

Bimoléculaire complémentation de fluorescence (BiFC) Dosage pour les interactions protéine-protéine dans les cellules d&#39;oignon Utilisation du pistolet à gènes Helios

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Chromatographie d&#39;affinité lipidique des vésicules médiée utilisant magnétique tri cellulaire activé (LIMACS): une nouvelle méthode pour analyser les interactions protéines-lipides

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Purification de Hsp104, une protéine Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

Un protocole pour Phage Display et Affinity sélection en utilisant appâts protéine recombinante

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

Identifier les interactions protéine-protéine dans<em&gt; Drosophila</em&gt; Têtes de Tandem Affinity Purification adultes (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

Identifier les sites d&#39;interaction protéine-protéine à l&#39;aide de peptides tableaux

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

L&#39;échelle du génome interaction protéine-protéine Le dépistage par Protein-fragment complémentation Assay (PCA) dans les cellules vivantes

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

Analyse de l’imagerie de la complémentation de luciférase dans les feuilles de Nicotiana benthamiana pour transitoirement déterminant la dynamique Interaction protéine-protéine

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...