Ce travail a été soutenu par des subventions des National Institutes of Health à VF (NS42599 et GM077569).

1. Préparation pour la réticulation <ol><li> Vous aurez besoin de la plaque d&#39;un nombre suffisant de cellules pour permettre l&#39;isolement de 500 pg de protéines par tube de réaction standard. Un tube unique peut être suffisante pour l&#39;identification des interacteurs putatif d&#39;une protéine d&#39;intérêt par immunoblot. Dans ce cas, perles du matériel lié peut être élue avec un tampon d&#39;échantillon SDS-PAGE (immunoprécipitation). Pour l&#39;analyse par spectrométrie de masse, le nombre de réactions norme devrait être augmenté d&#39;au moins dix fois et les complexes de protéines doit être élue par hors compétition avec un antigène peptidique reconnue par l&#39;anticorps utilisé (chromatographie d&#39;immunoaffinité). Cette stratégie permettra l&#39;isolement de complexes protéiques libres des immunoglobulines. </li><li> La densité cellulaire optimale pour la réticulation est comprise entre 75% et 90% de confluence. En conditions de haute confluence, les cellules ont tendance à s&#39;empiler les uns des autres, ce qui diminue l&#39;accessibilité aux réticulant cellulaire perméable à toutes les cellules. </li><li> Type de cellules et de l&#39;état expérimental devrait être étiqueté sur le fond de la plaque cellulaire. Nous avons régulièrement inclure des contrôles avec le véhicule seul. </li><li> Préparer toutes les solutions SAUF la solution DSP avant réticulation. <ol><li> Préparer un stock de tampon phosphate buffer saline avec CaCl 2 0,1 mM et CaCl2 1 mM (PBS / Ca / Mg) avant de commencer l&#39;expérience. L&#39;addition d&#39;ions calcium et magnésium est essentiel pour l&#39;adhérence des cellules à la plaque de la culture au cours de l&#39;expérience. Conserver à 4 ° C. </li><li> 50X cocktail complet d&#39;inhibiteur de protéase - 1 comprimé dissous dans 1ml eau Milli-Q. Conserver à -20 ° C. </li><li> 20% Triton X-100 Peser 10g de Triton X -100 et diluer dans le volume total de 50 ml d&#39;eau Milli-Q. Rock à 4 ° C pendant la nuit et conserver à 4 ° C jusqu&#39;à utilisation. Ne pas stocker plus de 1 mois. Utiliser des dilutions de ce stock de 20% à préparer les tampons de lyse et IP décrit ci-dessous. </li><li> 50X DSP Trempe solution 1M TRIS à pH 7,4. Stocker à température ambiante. </li><li> Préparer un tampon 10x Une solution. Tampon 10X A: <ul><li> 50 mL 1 M HEPES </li><li> 150 ml de NaCl 5 M </li><li> 10 ml 0,5 M EGTA </li><li> 250 uL 2 M MgCl 2 </li><li> Ajuster le pH à 7,4 </li><li> Amener le volume final à 500 ml </li></ul> Le Tampon 10X Une solution est diluée à 1X tampon A besoin que. Tampon 1x A est également utilisé dans le lysat et immuno-magnétique-précipitation tampons. Un tampon 1X <ul><li> 10 mM Hepes </li><li> 150 mM de NaCl </li><li> 1 mM d&#39;EGTA </li><li> 0,1 mM MgCl 2 </li><li> pH 7,4 </li></ul></li><li> Lysis Buffer 1X tampon A + 0,5% de Triton X-100. </li><li> Tampon précipitations immuno-magnétique (IP Buffer) 1X tampon A + 0,1% de Triton X-100. </li></ol></li></ol> 2. Préparation de la solution de réticulation <ol><li> Préparer la solution DSP immédiatement avant l&#39;application aux cellules. DSP est hautement hydrophobe et doit être dissous dans le DMSO avant dilution dans du PBS / Ca / Mg tampon. Dissoudre 40 mg de DSP dans 1 ml de DMSO. Cela fait une solution de 100 mM de DSP (Le poids moléculaire de 404,42 DSP est g / mol). </li><li> Chaud un volume approprié de PBS / Ca / Mg à 37 ° C afin de faciliter la dilution des DSP / DMSO dans du PBS. Volumes nécessaires pour les tailles de plaques assorties: 6-même plaque de 2 ml par puits Plaque de 10 cm de 10 ml par plaque 15 cm plaque de 20 ml par plaque </li><li> Ajouter 10 ul de solution stock DSP / DMSO pour chaque 1 mL d&#39;eau tiède PBS / Ca / Mg. Ajouter DSP / DMSO goutte à goutte la solution mère avec un mélange répété jusqu&#39;à ce que tous les DSP a dissous. Faire une solution de contrôle de 10 ul de DMSO ajoutée à chaque mL de PBS 1 / Ca / Mg. </li><li> Placez toutes les solutions de PBS / Ca / Mg dans un bain d&#39;eau glacée ne sont plus que 10 min. </li></ol> 3. Préparer les cellules de la réticulation <ol><li> Préparer un bain d&#39;eau glacée qui s&#39;adaptera à toutes les plaques de réticulation ou l&#39;incubation de contrôle du véhicule. </li><li> Prenez les plaques de l&#39;incubateur à 37 ° C et placez-les immédiatement dans le bain d&#39;eau glacée. </li><li> Laver les cellules deux fois avec PBS glacé / Ca / Mg. Utilisez le même volume que vous allez utiliser pour l&#39;incubation réticulant (voir section 2.2). </li><li> Retirer second lavage et ajouter le contrôle du véhicule soit ou d&#39;une solution tampon DSP réticulation. </li><li> Incuber dans la glace pendant deux heures. Vous devriez vérifier les plaques d&#39;environ toutes les vingt minutes pour s&#39;assurer que toutes les cellules sont couvertes par la solution. Vous remarquerez peut-être une petite quantité de DSP précipitent hors de la solution. Cela est normal. </li></ol> 4. L&#39;inactivation de la réaction du PSD <ol><li> Préparer une solution de trempe 1X DSP de 20 mM de Tris pH 7,4 dans du PBS / Ca / Mg (20 uL d&#39;une pH 7,4 M Tris pour chaque 1 mL de PBS / Ca / Mg). </li><li> Retirez le contrôle du véhicule et des solutions de réticulation. </li><li> Ajouter la solution de l&#39;inactivation glacée et incuber sur de la glace pendant 15 minutes. </li></ol> 5. Lyse cellulaire <ol><li> Durant l&#39;incubation trempe DSP, préparer le tampon de lyse cellulaire de 0,5% de Triton X-100 dans le tampon A avec cocktail inhibiteur de protéase complète. Ajouter 20 uL solution stock de 50X sur complètes pour chaque tampon lyse 1 ml. </li><li> Après l&#39;incubation trempe DSP 15 minutes, laver les cellules deux fois avec du PBS / Ca / Mg. </li><li> Ajouter un tampon de lyse complète + aux cellules et de rock à 4 ° C pendant 30 minutes. Certains ont suggéré des volumes de tampon de lyse pour les formats de plaques assorties: <ul><li> 6-même plaque de 0,5 ml par puits </li><li> Plaque de 10 cm de 1 ml par plaque </li><li> 15 cm plaque de 1 ml par plaque </li></ul></li><li> Après la lyse, d&#39;utiliser un grattoir à cellules pour recueillir les cellules de la plaque. Soyez sûr de garder lysats sur la glace afin de minimiser l&#39;activité de la protéase </li><li> Cellulaire Spin lysats pendant 15 minutes à 4 ° C dans une paillasse mini-centrifugeuse à grande vitesse (15.000 xg). </li><li> Pipeter le surnageant dans un nouveau tube. Jeter le culot. </li><li> Analyser les taux de protéines et de procéder à immunoprécipitation. </li></ol> 6. Préparation Perles précipitations immuno-magnétiques Note: Cette étape est généralement commencé directement après le début de l&#39;incubation de réticulation 2 heures. <ol><li> Combinez 30 uL de Dynal billes immunomagnétiques, 500 pi de tampon IP (1x tampon A + 0,1% de Triton X-100) et la quantité appropriée d&#39;antigène-anticorps spécifique à la vissable microtubes. Préparer sans anticorps et des tubes d&#39;anticorps non spécifique pour les contrôles négatifs. La quantité appropriée d&#39;anticorps doit être déterminée sur la base des antigènes individuels au cours des expériences distinctes. Étiqueter tous les tubes de PI. </li><li> Laisser les tubes IP à incuber dans un rocker fin plus-end à la température ambiante pendant deux heures. Alternativement, incuber les tubes IP avec l&#39;anticorps nuit à 4 ° le jour avant la reticulation. </li><li> Après l&#39;incubation, faire un rapide 10 secondes mini-centrifugation pour collecter les perles au fond du tube. </li><li> Faites glisser les tubes dans le support magnétique, qui va tirer les billes magnétiques loin du fond du tube. Avec les perles en toute sécurité hors du chemin, vous pouvez désormais facilement laver tout anticorps non liés. </li><li> Utiliser un embout d&#39;aspiration de petit diamètre, comme une pointe de chargement de gel, ou p200 astuce pour enlever le tampon d&#39;incubation et de tout anticorps non lié. Dès que le volume d&#39;incubation est retiré ajouter dans 1 ml de tampon de propriété intellectuelle. </li><li> Boucher le tube, doucement remettre en suspension les perles, et incuber tous les tubes IP à température ambiante pendant 5 minutes avec bout en bout au cours de rotation. </li><li> Répéter l&#39;étape de lavage de 5 minutes (étapes 6.3 à 6.6) fois de plus avec un millilitre de tampon fraîche IP. </li><li> Les tubes IP peuvent rester dans l&#39;incubation, laver jusqu&#39;à la lysat réticulé est prête à être ajoutée aux billes. </li></ol> 7. Incuber Lysat réticulé avec Immuno-magnétiques Perles <ol><li> Après le dernier lavage tour immuno-billes magnétiques, dans une mini-centrifugeuse pendant 10 secondes. Ensuite, passez à glisser les tubes dans le support magnétique. </li><li> Encore une fois, en utilisant une pointe d&#39;aspiration de petit diamètre, retirez le volume d&#39;incubation du tube. Immédiatement ajouter 500 ul de lysat cellulaire réticulé (en supposant lysat à 1 ug / ml) pour les tubes contenant l&#39;immuno-billes magnétiques. Si vous utilisez une concurrence peptide comme contrôle négatif, une quantité appropriée de peptide devraient être inclus à ce stade. Des expériences antérieures doivent être effectués afin de déterminer les conditions de concurrence peptide. Alternativement, vous pouvez avoir gratuitement lysat immuno-billes magnétiques comme contrôle négatif. Dans ce cas, ajouter immédiatement 500 de lysat Tampon (1x tampon A + 0,5% de Triton X-100) + complète sur les billes immuno-magnétiques. </li><li> Resuspendre doucement les perles. Perles en suspension doivent être incubées à 4 ° dans une rotation bout en bout au cours de 2 heures. </li></ol> 8. Lavez Lysat Unbound à partir de perles <ol><li> Une fois lysat a eu le temps d&#39;incubation suffisant, faire un rapide tour 10 secondes dans une mini-centrifugeuse. Faites ensuite glisser les tubes dans le support magnétique. Le support magnétique doivent être conservés dans un bain de glace pour le reste de l&#39;expérience. </li><li> En utilisant une pointe de petit aspirateur alésage, enlever tous lysat non lié. Immédiatement après toutes lysat non fixé est éliminé ajouter 1 mL de tampon de propriété intellectuelle. </li><li> Boucher le tube et délicatement remettre en suspension les perles. </li><li> Une fois que les billes sont remises en suspension répétez l&#39;étape de granulation billes (8,1). </li><li> Encore une fois Aspirer la totalité du volume d&#39;incubation et d&#39;ajouter immédiatement 1 ml de tampon IP, puis bouchon du tube. </li><li> Resuspendre doucement les billes en IP Buffer. Incuber perles resuspendues pendant 5 minutes à 4 ° à fin plus-end à bascule. </li><li> Répétez les étapes billes de lavage (8.4 à 8.6) 4 fois plus. </li></ol> 9. Dénaturer échantillon et recueillir des Perles <ol><li> Après tous les tubes de précipitations immuno-magnétiques ont été lavés, re-sédimenter les billes de (8,1) et les tubes glisser dans lasupport magnétique. </li><li> Protéines liées aux billes immuno-magnétiques sont enlevés dans des conditions dénaturantes. Aspirer le lavage IP Buffer dernière et retirer le tube de la propriété intellectuelle support magnétique. </li><li> Ajouter 1x tampon échantillon de gel dans le tube IP juste au-dessus du culot talon à la piscine des perles au fond du tube à nouveau. Le montant de tampon d&#39;échantillon dépend de la taille et du gel à être utilisé. Vous devrez peut-être taper doucement le fond du tube pour obtenir toutes les perles en suspension dans le tampon d&#39;échantillon de gel. </li><li> Répétez l&#39;opération de dénaturation (9,2 à 9,3) pour chaque tube IP. </li><li> Immuno-billes magnétiques en suspension dans du tampon échantillon de gel sont ensuite chauffées à 75 ° C pendant 5 minutes pour terminer le processus de dénaturation. </li><li> Une fois l&#39;échantillon a été dénaturé il peut être exécuté sur un gel SDS-PAGE pour western blot. Vidé immuno-billes magnétiques peuvent être re-sédimentées par centrifugation et retiré de l&#39;échantillon avec le support magnétique. </li></ol> 10. Élution des complexes réticulés avec des peptides antigéniques (chromatographie d&#39;immunoaffinité). <ol><li> Après tous les tubes de l&#39;immuno-magnétiques immunoprécipitation ont été lavés, re-sédimenter les billes de (8,1) et les tubes diapositives dans le support magnétique. Aspirer le surnageant et ajouter 10 ul de tampon A additionné avec le peptide antigénique reconnu par l&#39;anticorps utilisé pour immunoisolation de complexes protéiques. Vous devriez tester des concentrations allant de 10 à 200 uM pour l&#39;élution efficace. </li><li> Incuber les perles pendant 2 h à 4 ° C. </li><li> Mettez billes dans le support magnétique et soigneusement de récupérer les 10 ml de matériau élue. Sauvegarder cette surnageant. </li><li> Laver rapidement les billes avec un tampon A et jeter le lavage. Enregistrer les perles. </li><li> Ajouter tampon échantillon de gel au surnageant sauvés et des perles à une concentration de 1X. Incuber ces tubes à 75 ° C pendant 5 minutes. </li><li> Analyser des perles et éluat peptidique par SDS-PAGE. L&#39;éluat doit être libre d&#39;IgG à la fois par la protéine tache de SDS-PAGE ou par immunoblots (figure 1B). Ce matériau exempt d&#39;IgG est adapté à la spectrométrie de masse. </li></ol><img src="/files/ftp_upload/1855/1855fig1.jpg" alt="Figure 1" /> Figure 1. L&#39;isolement de l&#39;AP-3 complexes de protéines qui interagissent et des protéines membranaires. HEK293 (A) ou les cellules PC12 (B) ont été traités soit en présence de maîtrise du véhicule (DMSO, impair voies figure 1A) ou DSP (même ruelles Fig. 1A ou tous les voies dans la figure 1B). Clarification des extraits ont été incubées soit avec des perles seules (Fig. 1A, Lanes 3-4), les anticorps récepteurs de la transferrine (Fig. 1A, Lanes 5-6), ou AP-3 anticorps δ (Fig. 1A, Lanes 7-10; Fig . 1B, pistes 1-10). Des complexes immuns ont été éluées avec un tampon d&#39;échantillon SDS-PAGE (figure 1A), le tampon A, seul ou (Fig. 1B, pistes 1-2), ou le tampon A additionné de concentrations croissantes du peptide antigénique δ correspondant aux acides aminés 680 710 des humains δ-adaptine (NCBI: AAD03777; GI: 1923266) (Fig. 1B, les voies 3-10). Ce peptide se lie l&#39;anticorps δ. Après élution de peptides, les surnageants (S) et des perles (B) ont été analysés par immunoblot (Fig. 1B). AP-3, qui est détectée avec des anticorps contre le δ, β3, et σ3 sous-unités, coprécipités avec les facteurs suivants solubles: clathrine chaîne lourde (CCH), le BLOC-1 pallidin sous-unité, et la sous-unité VPS33B HOUBLON complexes, ainsi que la membrane des protéines phosphatidylinositol-4-kinase de type II alpha (PI4KIIα) et le transporteur de zinc 3 (ZnT3). Notez l&#39;absence de chaînes lourdes IgG de souris dans le peptide élué surnageant dans la Fig. 1B.

<ol>
	<li>Salazar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hermansky-Pudlak+syndrome+protein+complexes+associate+with+phosphatidylinositol+4-kinase+type+II+alpha+in+neuronal+and+non-neuronal+cells.">Hermansky-Pudlak syndrome protein complexes associate with phosphatidylinositol 4-kinase type II alpha in neuronal and non-neuronal cells.</a> J Biol Chem. 284, 1790-1802 (2009).</li><li>Lomant, A. J., Fairbanks, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+probes+of+extended+biological+structures:+synthesis+and+properties+of+the+cleavable+protein+cross-linking+reagent+[35S]dithiobis(succinimidyl+propionate.">Chemical probes of extended biological structures: synthesis and properties of the cleavable protein cross-linking reagent [35S]dithiobis(succinimidyl propionate.</a> J Mol Biol. 104, 243-261 (1976).</li><li>Xiang, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+DSP,+a+reversible+cross-linker,+to+fix+tissue+sections+for+immunostaining,+microdissection+and+expression+profiling.">Using DSP, a reversible cross-linker, to fix tissue sections for immunostaining, microdissection and expression profiling.</a> Nucleic Acids Res. 32, e185-e185 (2004).</li><li>Craige, B., Salazar, G., Faundez, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylinositol-4-Kinase+Type+II+Alpha+Contains+an+AP-3+Sorting+Motif+and+a+Kinase+Domain+that+are+both+Required+for+Endosome+Traffic.">Phosphatidylinositol-4-Kinase Type II Alpha Contains an AP-3 Sorting Motif and a Kinase Domain that are both Required for Endosome Traffic.</a> Mol Biol Cell. 19, 1415-1426 (2008).</li><li>Newell-Litwa, K., Seong, E., Burmeister, M., Faundez, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+and+non-neuronal+functions+of+the+AP-3+sorting+machinery.">Neuronal and non-neuronal functions of the AP-3 sorting machinery.</a> J Cell Sci. 120, 531-541 (2007).</li><li>Salazar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zinc+Transporter+ZnT3+interacts+with+AP-3+and+it+is+Targeted+to+a+Distinct+Synaptic+Vesicle+Subpopulation.">The Zinc Transporter ZnT3 interacts with AP-3 and it is Targeted to a Distinct Synaptic Vesicle Subpopulation.</a> Mol Biol Cell. 15, 575-587 (2004).</li><li>Trinkle-Mulcahy, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+specific+protein+interaction+partners+using+quantitative+mass+spectrometry+and+bead+proteomes.">Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes.</a> J Cell Biol. 183, 223-239 (2008).</li></ol>

DSP, une membrane perméable-, réticulant chimiquement réductibles à un bras espaceur de 12 A est utilisé pour stabiliser les interactions entre protéines passagères 1,2,3,4. Ici, nous témoigne de cette stratégie avec le complexe adaptateur AP-3 d&#39;un complexe protéine soluble qui reconnaît et trie les protéines membranaires dans des vésicules à partir des endosomes 5. AP-3 se lie sélectivement au transporteur de zinc ZnT3 et la kinase lipidique phosphatidylinositol-4-kinase de typ...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
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<td>Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P4417</td>
<td>Dissolve 1 tablet in 200 mL water; add MgCl2 to a final concentration of 1 mM and CaCl2 to a final concentration of 0.1 mM</td>
</tr>
<tr>
<td>Dithiobis (succinimidyl propionate) (DSP)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>22585</td>
<td>Moisture sensitive, store in air tight container 4&deg;C</td>
</tr>
<tr>
<td>Dimethyl sulphoxide (DMSO) Hybri-Max</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D2650</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100, SigmaUltra</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T9284</td>
<td>&nbsp;</td>
<tr>
<td>Dynabeads, Sheep anti-Mouse IgG</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>110.31</td>
<td>Beads are also available as sheep anti-rabbit</td>
</tr>
<tr>
<td>Dyna-Mag-2 magnet</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>123-21D</td>
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isolation of labile multi-protein complexes by in vivo controlled cellular cross-linking and immuno-magnetic affinity chromatography

La nature dynamique des machineries cellulaires est souvent construit sur des associations de protéines transitoires et / ou faible. Ces interactions faible affinité empêche méthodes rigoureuses pour l&#39;isolement et l&#39;identification des réseaux protéiques autour d&#39;une protéine d&#39;intérêt. L&#39;utilisation de réticulants chimiques permet la stabilisation sélective des interactions labiles, contournant ainsi les limites biochimiques pour la purification. Nous présentons ici un protocole prêtent des cellules en culture qui utilise un réticulant homobifonctionnels avec un bras espaceur de 12 Å, dithiobis (succinimidyl propionate) (DSP). DSP est clivée par réduction d&#39;un pont disulfure présent dans la molécule. Réticulation combinée avec la chromatographie d&#39;immunoaffinité de protéines d&#39;intérêt avec des billes magnétiques permet l&#39;isolement de complexes protéiques qui, autrement, ne résisterait pas à la purification. Ce protocole est compatible avec les techniques habituelles de western blot et il peut être étendu pour l&#39;identification des protéines par spectrométrie de masse 1. Stephanie A. Zlatic et Pearl V. Ryder ont contribué également à ce travail.

Watch this Scientific Journal Video about Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography at JoVE.com

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

La cellule perméables réticulant DSP [dithiobis (succinimidyl propionate)] stabilise les interactions transitoires et labiles<em&gt; In vivo</em&gt;, Qui permet leur isolement à l&#39;aide des techniques rigoureuses purification des protéines recombinantes complexes. Nous présentons ici une technique pour les cellules de réticulation en culture suivie de l&#39;isolement de complexes protéiques par immunoprécipitation.

Isolement du labiles complexes multi-protéiques par des In vivo Contrôlée Cellulaire réticulation et chromatographie d&#39;affinité immuno-magnétiques

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

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Biology

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

17.2K vues.  Emory University. La cellule perméables réticulant DSP [dithiobis (succinimidyl propionate)] stabilise les interactions transitoires et labiles In vivo, Qui permet leur isolement à l'aide des techniques rigoureuses purification des protéines recombinantes complexes. Nous présentons ici une technique pour les cellules de réticulation en culture suivie de l'isolement de complexes protéiques par immunoprécipitation.

Vidéo: Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, Parkinson's, and Huntington's diseases, hereditary amyotrophic lateral sclerosis, and type 2 diabetes. Owing to the metastable nature of these protein assemblies, it is difficult to assess their oligomer size distribution quantitatively using classical methods, such as electrophoresis, chromatography, fluorescence, or dynamic light scattering. Oligomers of amyloidogenic proteins exist as metastable mixtures, in which the oligomers dissociate into monomers and associate into larger assemblies simultaneously. PICUP stabilizes oligomer populations by covalent cross-linking and when combined with fractionation methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or size-exclusion chromatography (SEC), PICUP provides snapshots of the oligomer size distributions that existed before cross-linking. Hence, PICUP enables visualization and quantitative analysis of metastable protein populations and can be used to monitor assembly and decipher relationships between sequence modifications and oligomerization1. Mechanistically, PICUP involves photo-oxidation of Ru2+ in a tris(bipyridyl)Ru(II) complex (RuBpy) to Ru3+ by irradiation with visible light in the presence of an electron acceptor. Ru3+ is a strong one-electron oxidizer capable of abstracting an electron from a neighboring protein molecule, generating a protein radical1,2. Radicals are unstable, highly-reactive species and therefore disappear rapidly through a variety of intra- and intermolecular reactions. A radical may utilize the high energy of an unpaired electron to react with another protein monomer forming a dimeric radical, which subsequently loses a hydrogen atom and forms a stable, covalently-linked dimer. The dimer may then react further through a similar mechanism with monomers or other dimers to form higher-order oligomers. Advantages of PICUP relative to other photo- or chemical cross-linking methods3,4 include short (&#8804;1 s) exposure to non-destructive visible light, no need for pre facto modification of the native sequence, and zero-length covalent cross-linking. In addition, PICUP enables cross-linking of proteins within wide pH and temperature ranges, including physiologic parameters. Here, we demonstrate application of PICUP to cross-linking of three amyloidogenic proteins the 40- and 42-residue amyloid &beta;-protein variants (A&beta;40 and A&beta;42), and calcitonin, and a control protein, growth-hormone releasing factor (GRF).

Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides

Photo-induced cross-linking of unmodified proteins (PICUP) allows characterization of oligomer size distribution in metastable protein mixtures. We demonstrate application of PICUP to three representative amyloidogenic peptides the 40- and 42-residue forms of amyloid &beta;-protein, and calcitonin, and a control peptide growth-hormone releasing factor.

Photo-induite réticulation des protéines non modifiées (PICUP) appliqué à des peptides amyloïdogéniques

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, ...

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Biologie

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Identification des complexes protéiques avec la protéomique quantitative chez S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

Visualisation simple complexes moléculaires In Vivo Utilisation avancée de microscopie par fluorescence

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

ELIME (Enzyme Linked Immuno Magnetic électrochimique) Méthode de détection des mycotoxines

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

L&#39;imagerie in vivo chez la drosophile larves Intact au sub-cellulaire Résolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.

A general protocol for the use of isothermal titration calorimetry to monitor the binding thermodynamics for biological systems with moderate binding affinities is presented.

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

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction.  In biological ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Isolement de la chromatine par purification d&#39;ARN (CHIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Identification des complexes protéiques dans<em&gt; Escherichia coli</em&gt; En utilisant séquentielle Purification par affinité peptide en combinaison avec la spectrométrie de masse en tandem

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

La cartographie sur-AFM des propriétés élastiques de la paroi cellulaire: au tissu, cellulaire, et des résolutions Subcellular

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo suivi 4-Dimensional de hématopoïétiques cellules souches et progénitrices adultes dans la voûte du crâne de souris de moelle osseuse

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...