Il lavoro nel nostro laboratorio è sostenuto anche da finanziamenti del NIH, USDA Istituto Nazionale di Alimentazione e l&#39;Agricoltura, NSF, BARD, DOE, e BSF a VC

1. Espressione e l&#39;estrazione delle proteine <ol><li> Differenziale tag le proteine ​​da testare per la loro rilevazione. Etichetta la proteina di essere immobilizzato sulla membrana (ProIM) con un tag di dimensioni più grandi (ad esempio, GST), e il tag non condensato può essere utilizzato come controllo negativo immobilizzato (ProIMnc). Etichetta la proteina da usare come una sonda solubile (ProSol) sia con un grande o un tag di piccole dimensioni. Fusibile lo stesso tag di una proteina solubile appropriato controllo negativo (ProSOLnc) e includono nel test di interazione per confermare la specificità del legame rilevato. </li><li> Scegliere il sistema di espressione proteica di esprimere proteine ​​tag, vale a dire, E. coli, baculovirus, ecc, a seconda delle necessità di eventuali modifiche dopo traduzionali e le rese delle proteine. </li><li> Estratto ProIM e ProIM nc dall&#39;organismo di scelta (passo 1,2) con SDS-PAGE tampone di caricamento (20% glicerolo, 4% SDS, 20 mM Tris-HCl, pH6.8) contenente proteinasi cocktail di inibitori. Lascia la sospensione cellulare a temperatura ambiente per 15 minuti, aggiungere 0,5% di 2-mercaptoetanolo e fate bollire per 5 minuti. </li><li> Estratto ProSol e ProSol nc dall&#39;organismo di scelta (passo 1,2) utilizzando uno standard protocolli 16. Queste proteine ​​dovrebbero essere facilmente solubile nel buffer di legame (140 mM NaCl, 10 mM, Tris-HCl pH 7.4, 2 mM EDTA, 2 mM DTT, 1% di BSA, 0.1% Tween 20) utilizzato nel passaggio 4.1. </li><li> Determinare la concentrazione delle proteine ​​ricombinanti utilizzando un metodo standard, come Bio-Rad kit di analisi delle proteine. come il Bio-Rad kit di analisi delle proteine. Se estratto grezzo, piuttosto che le proteine ​​purificate, viene utilizzato per l&#39;analisi, stima la concentrazione della proteina di interesse in questo estratto attraverso la scansione densitometria della banda corrispondente su un SDS-poliacrilammide gel colorato con Coomassie Brilliant Blue R-250 e l&#39;utilizzo di note concentrazioni di BSA come riferimento. </li></ol> 2. Immobilizzazione di ProIM e ProIMnc sulla membrana <ol><li> Risolvere due serie di estratti, contenenti ciascuno 1 mg di ProIM e ProIM nc su un SDS-poliacrilammide gel secondo il protocollo standard 16. </li><li> Dopo l&#39;elettroforesi, posto il trasferimento del gel in 100 ml di buffer di trasferimento (60 mM glicina, 10 mM Tris, 0,0006% SDS, 20% MeOH) e incubare con leggera agitazione per 20 minuti a temperatura ambiente. </li><li> Electrotransfer le proteine ​​contenute nel gel ad una membrana di nitrocellulosa secondo il protocollo standard 16. </li></ol> 3. Re-piegatura di proteine ​​di membrana <ol><li> Dopo electrotransfer, incubare la membrana per 15 minuti in 15 ml di tampone A (30 mM Tris-HCl pH7.4, 0,05% Tween 20) e scuotendo con cautela per rimuovere residui di SDS. </li><li> Dopo aver svuotato con cura, trasferire la membrana a 25 ml di tampone di denaturazione (7 M guanidina, 2 mM EDTA, 50 mM DTT, 50 mM Tris-HCl pH 8,3). e incubare per 2 ore a temperatura ambiente con agitazione. (Nota: La membrana di nitrocellulosa diventa opaco quando incubati nel buffer di denaturazione). e incubare per 2 ore a temperatura ambiente con agitazione. </li><li> Trasferire la membrana di 25 ml di TBS (10 mM Tris-HCl, 150 mM NaCl, pH 7,4) e incubare con leggera agitazione per 5 min (Nota: durante questa fase, la membrana riacquista il suo originario colore bianco). </li><li> Trasferire la membrana a 25 ml di tampone vincolanti (vedi punto 1.2) e incubare a 4 ° C con agitazione per il pernottamento. </li></ol> 4. Sondaggio della ProIM da ProSol <ol><li> Tagliare la membrana in due strisce, entrambi contenenti ProIM e ProIM nc. </li><li> Rendere le soluzioni di ibridazione ProSol e ProSOLnc diluendo la preparazione con mcg 1-10 di entrambi ProSol o ProSOLnc in 10 ml di tampone fresco vincolanti. Trasferire ciascuna membrana nella soluzione di ibridazione ProSol o ProSOLnc e incubare per 1,5 ore a temperatura ambiente e scuotendo con cautela. </li><li> Rimuovere le membrane dalle soluzioni ibridazione e lavare tre volte per 15 minuti ciascuno in TBS. </li></ol> 5. Visualizzazione interazione proteina-proteina mediante immunoblotting <ol><li> Blocco della membrana con 2,5% di latte scremato in TBST (10 mM Tris-HCl, 140 mM NaCl, 0,05% Tween 20, pH 7,4) per 1 ora a temperatura ambiente. Diluire l&#39;anticorpo primario (anti-strepII anticorpo policlonale di coniglio) in 0,5% di latte scremato in TBST alla concentrazione consigliata dal produttore. </li><li> Posizionare le membrane bloccato nella soluzione di anticorpi, e incubate per 1 ora a temperatura ambiente o per la notte a 4 ° C con agitazione. </li><li> Sciacquare le membrane in 20 TBST ml per 15 minuti, e due volte per 5 minuti a temperatura ambiente con agitazione. </li><li> Diluire l&#39;anticorpo secondario (anti-IgG di coniglio) coniugati con perossidasi di rafano (HRP) in 0,5% di latte scremato in TBST alla concentrazione consigliata dal produttore. Posizionare le membrane in anticorpo secondariosoluzione e incubare per 1 ora a temperatura ambiente e scuotendo con cautela. </li><li> Sciacquare le membrane in 20 TBST ml per 15 minuti, e due volte per 5 minuti a temperatura ambiente con agitazione. Dopo l&#39;ultimo risciacquo con TBS, visualizzare le interazioni proteina-proteina utilizzando un substrato chemiluminescenza HRP (ad esempio, Millipore Immobilon occidentale chemiluminescente substrato HRP). </li><li> Sonda le membrane stesse con l&#39;anticorpo primario per il tag fusa ProIM, seguita da l&#39;anticorpo secondario appropriato, come descritto nei passaggi 5,1-5,4. Visualizza ProIM e ProIMnc come descritto al punto 5.5 per convalidare l&#39;identità delle bande ottenute nel test di sovrapposizione proteina di membrana (punto 5.5). Nella maggior parte dei casi, stripping non è necessario, perché il segnale ottenuto da questo passaggio è molto più forte segnale di residui ottenuti a partire dal punto 5.5. </li></ol> 6. Rappresentante dei risultati: L&#39;ANK-MP interazione è stata osservata da BiFC nelle cellule epidermiche tabacco (Figura 1A). Poiché MP è una proteina altamente insolubile se espresso in batteri o nelle piante, analisi delle proteine ​​della membrana sovrapposizione è stato adottato per convalidare questa interazione in vitro (Figura 1B). Estratti proteici contenenti 1 mg di GST-MP (ProIM) o non condensato GST (ProIM nc) sono stati risolti mediante SDS-poliacrilammide gel elettroforesi, seguita da electrotransfer una membrana di nitrocellulosa. Quando questi sono stati sondati ProIMs solubile ANK-strepII (ProSol), GST-MP, ma non non condensato GST, esposti vincolante (Figura 1B, corsie 1, 2, confrontare corsie 5, 6). Inoltre, quando lo stesso insieme di ProIMs sono stati sondati con un ProSOLnc non correlate, cioè, Arabidopsis chinasi NADH citoplasmatico tag strepII (NADH3-strepII), non vincolante, non è stato osservato, dimostrando ulteriormente la specificità del ANK-MP interazione (Figura 1B, corsie 3, 4). <img src="/files/ftp_upload/2961/2961fig1.jpg" alt="Figura 1"/> Figura 1. Legame specifico di tabacco ANK a TMV MP in vivo e in vitro. (A) ANK-MP interazione in vivo le cellule epidermiche tabacco come rilevato dal BiFC. Forte segnale YFP è stato ricostruito quando MP e ANK, fusa al C-terminale e N-terminale metà del YFP, rispettivamente, sono stati coespressi nell&#39;epidermide tabacco seguenti microbombardment dei loro geni codifica. Questo segnale BiFC accumulata nel puncta alla periferia delle cellule, che sono diagnostici di plasmodesmi 15. (B) ANK-MP interazione in vitro, come rilevato dal test di sovrapposizione della membrana proteica. Estratti proteici contenenti 1 mg di GST-MP (ProIM) o non condensato GST (ProIMnc) sono stati risolti in un 15% di SDS-poliacrilammide gel, seguita da electrotransfer su una membrana di nitrocellulosa. Il GST-MPProIM e GSTProIMnc sono stati incubati con 0,5 mg / ml di ANK-strepII (ProSol), e ANK legame è stato rilevato dal sondaggio della membrana con anticorpi anti-strepII anticorpo policlonale di coniglio, seguita da anti-coniglio di anticorpi IgG + M secondario coniugato a HRP (corsie 1 e 2). Né GST-MPProIM né GSTProIMnc interagito con una proteina non correlate, Arabidopsis chinasi citoplasmatica NADH, fusa al tag strepII (ProSOLnc, corsie 3 e 4). L&#39;identità della band osservato in questa analisi è stata confermata dal sondaggio la membrana con anticorpi anti-GST (corsie 5 e 6). Quando la membrana è stata trattata con tampone di denaturazione senza essere lavati con tampone A, il legame della GST-MP per ANK è perso, mentre le proteine ​​non identificate contenute nel GST-MP e gli estratti proteici GST contining reagito con ANK-strepII, dimostrando l&#39;importanza del passo 3,1 prima del processo di denaturazione (corsie 7 e 8).

<ol>
	<li>Hu, C. D., Chinenov, Y., Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+interactions+among+bZIP+and+Rel+family+proteins+in+living+cells+using+bimolecular+fluorescence+complementation.">Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation.</a> Mol Cell. 9, 789-798 (2002).</li><li>Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+localization+of+interacting+proteins+by+bimolecular+fluorescence+complementation+in+planta.">Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta.</a> J Mol Biol. 362, 1120-1131 (2006).</li><li>Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+implementation+of+bimolecular+fluorescence+complementation+(BiFC)+assays+for+the+visualization+of+protein+interactions+in+living+cells.">Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells.</a> Nat Protoc. 1, 1278-1286 (2006).</li><li>Shyu, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+protein+interactions+in+living+Caenorhabditis+elegans+using+bimolecular+fluorescence+complementation+analysis.">Visualization of protein interactions in living Caenorhabditis elegans using bimolecular fluorescence complementation analysis.</a> Nat Protoc. 3, 588-596 (2008).</li><li>Citovsky, V., Gafni, Y., Tzfira, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localizing+protein-protein+interactions+by+bimolecular+fluorescence+complementation+in+planta.">Localizing protein-protein interactions by bimolecular fluorescence complementation in planta.</a> Methods. 45, 196-206 (2008).</li><li>Ohad, N., Shichrur, K., Yalovsky, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+analysis+of+protein-protein+interactions+in+plants+by+bimolecular+fluorescence+complementation.">The analysis of protein-protein interactions in plants by bimolecular fluorescence complementation.</a> Plant Physiol. 145, 1090-1099 (2007).</li><li>Zamyatnin, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+the+integral+membrane+protein+topology+in+living+cells.">Assessment of the integral membrane protein topology in living cells.</a> Plant J. 46, 145-154 (2006).</li><li>Citovsky, V., Knorr, D., Schuster, G., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+P30+movement+protein+of+tobacco+mosaic+virus+is+a+single-strand+nucleic+acid+binding+protein.">The P30 movement protein of tobacco mosaic virus is a single-strand nucleic acid binding protein.</a> Cell. 60, 637-647 (1990).</li><li>Heinlein, M., Epel, B. L., Padgett, H. S., Beachy, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+tobamovirus+movement+proteins+with+the+plant+cytoskeleton.">Interaction of tobamovirus movement proteins with the plant cytoskeleton.</a> Science. 270, 1983-1985 (1995).</li><li>Tomenius, K., Clapham, D., Meshi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+by+immunogold+cytochemistry+of+the+virus+coded+30+K+protein+in+plasmodesmata+of+leaves+infected+with+tobacco+mosaic+virus.">Localization by immunogold cytochemistry of the virus coded 30 K protein in plasmodesmata of leaves infected with tobacco mosaic virus.</a> Virology. 160, 363-371 (1987).</li><li>Ding, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+plasmodesmata+are+specific+sites+of+localization+of+the+tobacco+mosaic+virus+movement+protein+in+transgenic+tobacco+plants.">Secondary plasmodesmata are specific sites of localization of the tobacco mosaic virus movement protein in transgenic tobacco plants.</a> Plant Cell. 4, 915-928 (1992).</li><li>Meshi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+the+30+kd+protein+of+tobacco+mosaic+virus:+involvement+in+cell-to-cell+movement+and+dispensability+for+replication.">Function of the 30 kd protein of tobacco mosaic virus: involvement in cell-to-cell movement and dispensability for replication.</a> EMBO J. 6, 2557-2563 (1987).</li><li>Wolf, S., Deom, C. M., Beachy, R. N., Lucas, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Movement+protein+of+tobacco+mosaic+virus+modifies+plasmodesmatal+size+exclusion+limit.">Movement protein of tobacco mosaic virus modifies plasmodesmatal size exclusion limit.</a> Science. 246, 377-379 (1989).</li><li>Waigmann, E., Lucas, W. J., Citovsky, V., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+functional+assay+for+tobacco+mosaic+virus+cell-to-cell+movement+protein+and+identification+of+a+domain+involved+in+increasing+plasmodesmal+permeability.">Direct functional assay for tobacco mosaic virus cell-to-cell movement protein and identification of a domain involved in increasing plasmodesmal permeability.</a> Proc. Natl. Acad. Sci. USA. 91, 1433-1437 (1994).</li><li>Ueki, S., Spektor, R., Natale, D. M., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ANK,+a+host+cytoplasmic+receptor+for+the+Tobacco+mosaic+virus+cell-to-cell+movement+protein,+facilitates+intercellular+transport+through+plasmodesmata.">ANK, a host cytoplasmic receptor for the Tobacco mosaic virus cell-to-cell movement protein, facilitates intercellular transport through plasmodesmata.</a> PLoS Pathog. 6, e1001201-e1001201 (2010).</li><li>Coligan, J. E., Dunn, B. M., Ploegh, H. L., Speicher, D. W., Wingfield, P. T., Taylor, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (2011).</li><li>Tzfira, T., Vaidya, M., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VIP1,+an+Arabidopsis+protein+that+interacts+with+Agrobacterium+VirE2,+is+involved+in+VirE2+nuclear+import+and+Agrobacterium+infectivity.">VIP1, an Arabidopsis protein that interacts with Agrobacterium VirE2, is involved in VirE2 nuclear import and Agrobacterium infectivity.</a> EMBO J. 20, 3596-3607 (2001).</li><li>Chen, M. H., Sheng, J., Hind, G., Handa, A., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+the+tobacco+mosaic+virus+movement+protein+and+host+cell+pectin+methylesterases+is+required+for+viral+cell-to-cell+movement.">Interaction between the tobacco mosaic virus movement protein and host cell pectin methylesterases is required for viral cell-to-cell movement.</a> EMBO J. 19, 913-920 (2000).</li></ol>

Nessun conflitto di interessi dichiarati.

Questo approccio è adatto per testare interazioni proteina-proteina tra le combinazioni di proteine, quando almeno uno dei quali le proteine ​​è facilmente solubile nel buffer vincolante, ed è stato applicato con successo ad altra combinazione di proteine ​​17,18. Il iInteractions tra le proteine ​​che sono sia insolubile in queste condizioni non possono essere testati da questo protocollo. Inoltre, il successo di ripiegamento ProIM è fondamentale per l&#39;analisi...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Proteine ​​test kit </td><td> Bio-Rad </td><td> 500-0001 </td></tr><tr><td> Inibitore della proteasi cocktail </td><td> SIGMA </td><td> S8820 </td></tr><tr><td> Mini-PROTEAN sistema </td><td> Bio-Rad </td><td> 165-8000 </td></tr><tr><td> Semi-secco western blotting SD electrotransfer sistema </td><td> Bio-Rad </td><td> 170-3940 </td></tr><tr><td> Anti-IgG di coniglio coniugato con perossidasi di rafano </td><td> GenScript </td><td> A00098 </td></tr><tr><td> Anti-GST coniglio anticorpo policlonale </td><td> GenScript </td><td> A00097 </td></tr><tr><td> Anti-strepII </td><td> GenScript </td><td> A00626 </td></tr><tr><td> Biotrace, NT nitrocellulosa membrana trasferimento </td><td> Pall scientifico </td><td> 27377-000 </td></tr><tr><td> Immobilon occidentale HRP substrato chemiluminescente </td><td> Millipore </td><td> WBKL S0 050 </td></tr></tbody></table>

protein membrane overlay assay: a protocol to test interaction between soluble and insoluble proteins in vitro

Convalida interazioni tra proteine ​​diverse è fondamentale per l&#39;esame delle loro funzioni biologiche a livello molecolare. Ci sono diversi metodi, sia in vitro che in vivo, per valutare legame con le proteine, e almeno due metodi che integrano le carenze di ogni altro dovrebbe essere condotta di acquisire le conoscenze affidabili. Per un test in vivo, la complementazione bimolecolare fluorescenza (BiFC) saggio rappresenta l&#39;approccio più popolare e meno invasivo che permette di rilevare l&#39;interazione proteina-proteina all&#39;interno delle cellule viventi, così come individuare la localizzazione intracellulare del 1,2 interagendo proteine. In questo saggio, non fluorescente metà N-e C-terminale della GFP o di sue varianti si fondono alle proteine ​​testate, e quando le due proteine ​​di fusione sono riuniti a causa delle interazioni delle proteine ​​testate &#39;, il segnale fluorescente viene ricostituito 3-6 . Perché il suo segnale è facilmente rilevabile mediante microscopia in epifluorescenza e confocale, BiFC è emersa come un potente strumento di scelta tra i biologi cellulari per lo studio di interazioni proteina-proteina nelle cellule viventi 3. Questo test, tuttavia, possono a volte produrre risultati falsi positivi. Per esempio, il segnale fluorescente può essere ricostituito da due frammenti GFP disposte, per quanto 7 nm l&#39;una dall&#39;altra a causa di chiudere imballaggio in un piccolo vano subcellulare, piuttosto che a causa di interazioni specifiche 7. A causa di queste limitazioni, i risultati ottenuti dalle tecnologie di imaging cellulare dal vivo deve essere confermata da un approccio indipendente, basato su un principio diverso per rilevare le interazioni delle proteine. Co-immunoprecipitazione (Co-IP) o glutatione transferasi (GST) pull-down test rappresentano tali metodi alternativi che sono comunemente utilizzati per analizzare interazioni proteina-proteina in vitro. Tuttavia, iIn questi test, però, le proteine ​​testato deve essere facilmente solubile nel buffer che supportsused per la reazione di legame. Pertanto, le interazioni specifiche che coinvolgono una proteina insolubile che non può essere valutata da queste tecniche. Qui, ci illustrano il protocollo per il saggio sovrapposizione proteina di membrana vincolante, che aggira questa difficoltà. In questa tecnica, l&#39;interazione tra proteine ​​solubili e insolubili in modo attendibile testato perché una delle proteine ​​è immobilizzato su una matrice di membrana. Questo metodo, in combinazione con esperimenti in vivo, come BiFC, fornisce un approccio affidabile per studiare e caratterizzare le interazioni tra fedelmente proteine ​​solubili e insolubili. In questo articolo, vincolante tra virus del mosaico del tabacco (TMV) proteina movimento (MP), che esercita funzioni multiple durante virale cellula-cellula trasporto 8-14, e un impianto di Interactor recentemente identificato cellulari, tabacco ankyrin ripetuta contenenti proteine ​​(ANK ) 15, si dimostra con questa tecnica.

Watch this Scientific Journal Video about Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro at JoVE.com

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Test interazione proteina-proteina è indispensabile per la dissezione di funzionalità delle proteine. Qui, introdurre un<em&gt; In vitro</em&gt; Proteina-proteina saggio vincolante per sondare una membrana-proteina immobilizzato con una proteina solubile. Questa analisi fornisce un metodo affidabile per verificare l&#39;interazione tra una proteina insolubile e una proteina in soluzione.

Proteine ​​di membrana Overlay saggio: un protocollo di prova interazione tra proteine ​​solubili e insolubili In vitro</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

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Biology

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

22.0K Views.  State University of New York. Test interazione proteina-proteina è indispensabile per la dissezione di funzionalità delle proteine. Qui, introdurre un In vitro Proteina-proteina saggio vincolante per sondare una membrana-proteina immobilizzato con una proteina solubile. Questa analisi fornisce un metodo affidabile per verificare l'interazione tra una proteina insolubile e una proteina in soluzione.

Video: Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

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.

La colorazione delle proteine ​​in gel con Coomassie G-250, senza solventi organici acido acetico e

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

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Biologia

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)

Biomolecolare fluorescenza complementazione (BiFC) Assay per interazione proteina-proteina nelle cellule Cipolla Utilizzando la Helios Gun Gene

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

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

Un liquido fase di cattura test affinità utilizzando biglie magnetiche per studiare interazioni proteina-proteina: il poliovirus-Nanobody Esempio

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

L&#39;utilizzo di un robot per High-throughput cristallizzazione delle proteine ​​di membrana in mesofasi lipidici

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

In vitro metilazione Assay per studiare proteine ​​Arginina Metilazione

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

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).

Genome-wide proteina-proteina interazione Screening per Protein-frammento Complementation Assay (PCA) in cellule viventi

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

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Green Fluorescent Protein-base Espressione Proiezione di proteine ​​di membrana in<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

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.

Analisi di Imaging di complementazione di luciferase in foglie di Nicotiana benthamiana per transitoriamente determinare dinamiche di interazione proteina-proteina

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

Determination of Tripartite Interaction between Two Monomers of a MADS-box Transcription Factor and a Calcium Sensor Protein by BiFC-FRET-FLIM Assay

Protein-protein interactions are an integral part of all biological processes in the cells as they play a crucial role in regulating, maintaining, and amending cellular functions. These interactions are involved in a wide range of phenomena such as signal transduction, pathogen response, cell-cell interactions, metabolic and developmental processes. In the case of transcription factors, these interactions may lead to oligomerization of subunits, sequestering in specific subcellular contexts such as the nucleus, cytoplasm, etc., which, in turn, might have a more profound effect on the expression of the downstream genes. Here, we demonstrate a methodology to visualize in vivo tripartite interaction using Bimolecular Fluorescence Complementation (BiFC) based F&#246;rster Resonance Energy Transfer (FRET) involving Fluorescence Lifetime Imaging (FLIM). Two of the proteins selected for this demonstration interact as BiFC partners, and their reconstituted fluorescence activity is used to assay FRET-FLIM with the third partner. Four to five-week-old growth-chamber-grown Nicotiana benthamiana plants have been used as the model plant system for this demonstration.

Here we present, a method to visualize ternary complex formation between three protein partners using fluorescent-tagged proteins by BiFC based FRET-FLIM assay. This method is valuable for studying protein-protein interaction complexes in vivo.

Determinazione dell'interazione tripartita tra due monomeri di un fattore di trascrizione MADS-box e una proteina sensore di calcio mediante saggio BiFC-FRET-FLIM

Protein-protein interactions are an integral part of all biological processes in the cells as they play a crucial role in regulating, maintaining, and ...

Produzione e imaging di vescicole ricombinanti piene di proteine da E. coli

Optimized Production and Analysis of Recombinant Protein-Filled Vesicles from E. coli

This innovative system, using a short peptide tag, that exports multiple recombinant proteins in membrane bound vesicles from E. coli, provides an effective solution to a range of problems associated with bacterial recombinant protein expression. These recombinant vesicles compartmentalise proteins within a micro-environment that facilitates the production of otherwise challenging, toxic, insoluble, or disulfide-bond containing proteins from bacteria. Protein yield is increased considerably when compared to typical bacterial expression in the absence of the vesicle-nucleating peptide tag. The release of vesicle-packaged proteins supports isolation from the culture medium&#160;and permits long-term active protein storage. This technology gives rise to increased yields of vesicle-packaged, functional proteins for simplified downstream processing for a diverse range of applications from applied biotechnology to discovery science and medicine. In the present article and the associated video, a detailed protocol of the method is provided, which highlights key steps in the methodology to maximize recombinant protein-filled vesicle production.

Autore in primo piano: La produzione di proteine ricombinanti 

The present protocol describes a detailed method for the bacterial production of recombinant proteins, including typically insoluble or disulfide-bond containing proteins, packaged inside extracellular membrane-bound vesicles. This has the potential to be applied to versatile areas of scientific research, including applied biotechnology and medicine.

Proteine ​​di membrana Overlay saggio: un protocollo di prova interazione tra proteine ​​solubili e insolubili In vitro

Proteine di membrana Overlay saggio: un protocollo di prova interazione tra proteine solubili e insolubili In vitro