This work was funded by the Wellcome Trust (WT098051).

<ol>
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The authors have nothing to disclose.

Qui, si descrive l&#39;utilizzo di purificazione di affinità seguita da elettroforesi su gel nativo blu in combinazione con la spettrometria di massa quantitativa per risolvere complessi proteici. Questo approccio offre un metodo di svelare elenchi interazione proteina unidimensionali in assiemi proteina funzionale. Dimostriamo il metodo basato sull&#39;utilizzo di proteine ​​epitopo-tag. Tuttavia, se una linea cellulare che esprime una proteina tag non è disponibile, in alternativa potrebbe essere quella di utilizzare gli anticorpi contro la proteina di interesse, purché vi sia un peptide a disposizione per raggiungere eluizione competitiva nativo. La quantità di materiale di partenza e la quantità di perline potrebbe essere necessario modificare a seconda del livello di espressione della proteina bersaglio. Noi di solito effettuare questo protocollo con 2-5 x 10 8 cellule materiale di partenza, e questo è sufficiente anche per le proteine a basso livello di espressione. La composizione del tampone di lisi dovrebbe essere scelto empiricamente per ottenerevicino alla completa solubilizzazione dell&#39;esca. Questo potrebbe essere difficile per alcuni tipi di proteine, in particolare legame cromatina o proteine ​​di membrana. Opzioni alternative includono l&#39;aumento della quantità di sale, purché il complesso proteina in esame è stabile in alto sale, o utilizzando sonicazione e / o il trattamento nucleasi per cromatina proteine leganti 20, 29. Nel caso delle proteine di membrana, il passaggio detersivo DDM o digitonina può essere consigliabile 30. Nel caso di legame al DNA proteine, è utile includere un nucleasi come benzonasi durante la fase di purificazione 20. La rimozione completa di acidi nucleici assicura che le interazioni rilevate si verificano tra le proteine ​​e non sono mediati da DNA. Un elemento fondamentale da considerare quando si utilizza questo approccio è la stabilità del complesso sotto inchiesta. La procedura è lunga e può comportare preserving proteina overnight complesso. Abbiamo raggiunto un buon successo con due complessi di rimodellamento della cromatina (D. Bode e M. Pardo, dati non mostrati), ma questo deve essere valutato. La fase di purificazione per affinità può essere abbreviato se necessario. tecniche alternative che consentono frazionamento nativo, come cromatografia di esclusione dimensionale, sono stati ampiamente utilizzati per oltre 50 anni per caratterizzare complessi proteici. Noi e altri hanno dimostrato che la risoluzione del blu PAGE nativa è superiore a quella ottenuta mediante cromatografia di esclusione dimensionale 20, 31, 32, 33. Un altro vantaggio di blu PAGINA nativa è che non richiede sistemi di cromatografia, che sono costosi, ma piuttosto utilizza proteine ​​attrezzature elettroforetico che è molto diffusa nei laboratori. In termini di hands-on tempo, questo metodo non comporta più lavoro rispetto al tradizionale geLC-MS / MS unAPPROCCIO o offline cromatografica frazionamento. Tuttavia, come la maggior parte delle tecniche di frazionamento fanno, ha un limite alla loro risoluzione. Complessi che sono molto omogenei o chiudere in massa e la forma può essere oltre la risoluzione offerta dal blu PAGE nativa, e quindi il protocollo come riportato qui potrebbe non essere universalmente riuscito a risolvere complessi distinti con subunità condivisi. È incoraggiante, abbiamo avuto successo nel separare due complessi tetrameriche molto simili che condividono tre subunità (M. Pardo, manoscritto in preparazione). Poiché spettrometria di massa è diventata sempre più sensibile, anche piccole quantità di interattori e contaminanti non specifici possono essere rilevati in campioni di AP-MS. L&#39;approccio qui presentato potrebbe aiutare nel discriminare interattori reali dalla contaminazione di fondo nella purificazione di affinità focalizzando l&#39;attenzione sulle proteine ​​con picchi di migrazione che coincidono con picchi di migrazione esche a più alto peso molecolare di quello dile proteine ​​monomeriche. Diversi gruppi hanno utilizzato tecniche di frazionamento seguite da proteine correlazione profilatura a delineare complessi proteici in scala cellulare senza bisogno di precedente isolamento 10, 11, 12, 34. Tuttavia, ciò può comportare il mancato rilevamento interazioni sub-stechiometrico. L&#39;incorporazione di una fase di arricchimento attraverso la purificazione di affinità può aiutare a superare questo. L&#39;approccio qui descritto dovrebbe essere generalmente utile per esplorare la topologia di complessi proteici e disfare le molteplici complessi una data proteina partecipa nello stesso contesto cellulare. La strategia è semplice e suscettibili di laboratori che non possono avere costose apparecchiature di frazionamento cromatografico per risolvere complessi proteici.

Nelle cellule, maggior parte delle proteine ​​svolgere le loro funzioni attraverso transitori interazioni proteina-proteina o formando complessi proteici stabili. Caratterizzare le interazioni delle proteine ​​è fondamentale per i processi cellulari completamente comprensione. purificazione di affinità in combinazione con la spettrometria di massa (AP-SM) è una delle strategie più comunemente applicati per identificare le interazioni proteina nativa. Miglioramenti significativi nella capacità dello strumento raggiunti negli ultimi dieci anni hanno reso questo approccio estremamente potente. E &#39;importante notare che le interazioni identificate da esperimenti di AP-MS comprendono una miscela di associazioni dirette e indirette tra esche e prede. Inoltre, spesso proteine ​​partecipano a numerosi complessi diversi all&#39;interno dello stesso contesto cellulare, che potrebbero avere diversi ruoli biologici, e quindi le interattori identificati da AP-MS possono rappresentare una miscela di complessi proteici distinte o entità funzionali. Non è possibile derivaretali informazioni topologica a priori dalle liste mono-dimensionale di proteine generati da semplici esperimenti AP-MS. Tuttavia, la tecnica può essere sfruttata ulteriormente per definire l&#39;architettura di complessi proteici combinandolo con uno o più metodi per risolvere questi gruppi. Al fine di risolvere la topologia delle interazioni proteiche individuati attraverso AP-MS, sono stati applicati diverse strategie. Un approccio è quello di eseguire esperimenti iterativi AP-MS utilizzando le prede identificati in un precedente round di esperimenti come esche 1. Anche se molto informativo, questo è piuttosto un compito alta intensità di lavoro sia sperimentalmente e analiticamente. Proteina reticolazione in combinazione con la spettrometria di massa è sempre più utilizzata per ricavare informazioni topologiche su complessi proteici 2, 3, 4, 5. Tuttavia, computatioanalisi nale peptidi reticolati rimane ancora un compito impegnativo e quindi è il collo di bottiglia nel flusso di lavoro. L&#39;avvento di MS-scindibili reagenti di reticolazione dovrebbe facilitare la mappatura dei residui di amminoacidi che si trovano in prossimità di proteine interagenti 6, 7. Un&#39;altra alternativa è quella di combinare la purificazione per affinità con le precedenti tecniche di separazione ortogonali 8, 9. Frazionamento cromatografico mediante gel filtrazione o scambio ionico, o saccarosio frazionamento gradiente sono recentemente stato usato in combinazione con la spettrometria di massa quantitativa per descrivere complessi multiproteici a livello di tutto il sistema, by-passando la fase di complesso di isolamento 10, 11, 12, 13. Blu gel di poliacrilammide nativo elettroforesi (BN-PAGE) è stato ampiamente applicato aindagare le interazioni proteina nativa, in genere quelli che coinvolgono membrana mitocondriale complessi proteici 14. Questa tecnica di separazione è stato recentemente utilizzato in combinazione con quantificazione della proteina priva di etichetta e correlazione profilatura, non solo su complessi mitocondriali 15, 16, 17, ma anche per dipanare altri complessi proteici da cellule intere 18, 19. Abbiamo ipotizzato che la combinazione di purificazione di affinità con i successivi approcci frazionamento nativi e MS quantitativa dovrebbe fornire una strategia utile per risolvere più assembly proteina contenente una particolare proteina. Qui si descrive un metodo che combina nucleo purificazione per affinità epitopo-based con blu nativo elettroforesi su gel di poliacrilammide dei complessi isolati, seguita da sp massa quantitativaectrometry e proteine ​​correlazione profilatura, per risolvere i più assembly una proteina potrebbe essere coinvolta. Impieghiamo cellule staminali embrionali di topo in cui una proteina di interesse fusa ad un epitopo è espresso dal locus endogeno per ottenere vicino all&#39;abbondanza fisiologica e garantire nativo efficiente isolamento complesso. Questo approccio svela i molteplici interazioni proteina impegna in, risolverli in assiemi distinti sulla base dei loro profili di correlazione, mentre non richiede più lavoro rispetto ai tradizionali geLC-MS 20.

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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Protein G Dynabeads</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">10003D</td>
			<td style="width:452px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">FLAG antibody M2</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">F1804</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Tween-20, protein grade, 10% solution</td>
			<td style="width:285px;">Millipore</td>
			<td style="width:220px;">655206</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Dimethyl pimelimidate</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">80490</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">0.2 M Triethanolamine buffer, pH 8.2</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">T0449</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">3x FLAG peptide</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">F4799</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Vivaspin 5K NMWCO, PES</td>
			<td style="width:285px;">Sartorius</td>
			<td style="width:220px;">VS0112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">NativePAGE 3-12% Bis-Tris gel</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">BN1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">20x NativePAGE Running Buffer</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">BN2001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">20x NativePAGE Cathode Additive</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">BN2002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">4x NativePAGE sample loading buffer</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">BN2003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">NativeMARK</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">LC0725</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NativePAGE 5% G-250 sample additive&#160;</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">BN2004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Nunc conical bottom 96-well plate, polypropylene</td>
			<td style="width:285px;">Thermo</td>
			<td style="width:220px;">249944</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Multiscreen HTS 96-well filtration system</td>
			<td style="width:285px;">Thermo</td>
			<td style="width:220px;">MSDVN6550</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Nunc 96-well cap natural</td>
			<td style="width:285px;">Thermo</td>
			<td style="width:220px;">276002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">TCEP (Tris(2-carboxyethyl)phosphine hydrochloride)</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">646547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Iodoacetamide</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">I1149</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Acetonitrile, HPLC grade</td>
			<td style="width:285px;">Fisher Scientific</td>
			<td style="width:220px;">A/0627/17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Trypsin, sequencing grade</td>
			<td style="width:285px;">Roche</td>
			<td style="width:220px;">11418475001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Software</td>
			<td style="width:285px;"></td>
			<td style="width:220px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">MaxQuant (software)</td>
			<td style="width:285px;">N.A.</td>
			<td style="width:220px;">N.A.</td>
			<td style="width:452px;">http://www.biochem.mpg.de/5111795/maxquant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Perseus (software)</td>
			<td style="width:285px;">N.A.</td>
			<td style="width:220px;">N.A.</td>
			<td style="width:452px;">http://www.biochem.mpg.de/5111810/perseus</td>
		</tr>
	</tbody>
</table>

resolving affinity purified protein complexes by blue native page and protein correlation profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Il flusso di lavoro della purificazione di affinità Blu nativa delle proteine Correlazione profilatura mediante spettrometria di massa (ABC-MS) strategia è rappresentata in figura 7. complessi proteici nativi intorno ad una proteina di interesse sono isolati da purificazione per affinità utilizzando anticorpi contro un tag epitopo (in questo caso FLAG) ed eluizione competitivo. I complessi si risolvono con BN-PAGE, e tutta la corsia di gel viene asportato in 48 sezioni, e preparati per il fucile da caccia LC-MS / MS. Quantitative informazioni MS viene utilizzato per generare un profilo di migrazione per ogni proteina identificata attraverso la separazione nativo blu. Proteine ​​che interagiscono per formare un distinti profili migratori simili visualizzazione complesso con picchi sovrapponibili. Quando una proteina di interesse partecipa a più di un assemblaggio, picchi multipli si osservano nel suo profilo migrazione, date le sotto-complessi sono nel potere di risoluzione del gel nativo blu. Un confronto sistematico del migprofili razione può essere ottenuto mediante correlazione proteina profilatura usando clustering gerarchico. Dendrogramma e l&#39;intensità dei picchi per tutte le frazioni vengono visualizzati in una mappa termica, facilitando l&#39;identificazione di proteine interagenti che appartengono a distinti complessi proteici (Figura 8). Abbiamo usato questa strategia per analizzare i partner che interagiscono di MTA2, una subunità nucleo della cromatina nurd rimodellamento complesso 20. Come mostrato in figura 9, il profilo di migrazione MTA2 visualizzata due picchi di intensità distinti tra 700 kDa e 1,2 MDa, con il picco di massa inferiore visualizzazione maggiore abbondanza. Altre subunità nucleo nurd, tra mta1 / 3, HDAC1 / 2 e Mbd3, mostravano modello separazione identici, sebbene i picchi per alcune delle subunità, cioè Chd4, Gatad2a / b e Rbbp4 / 7, avevano la distribuzione abbondanza inversa (Figura 9A, B). Al contrario, il profilo di Cdk2ap1, un fattore di regolazione che recluta il complesso nurd di promotori genici Wnt 23, visualizzato solo il picco di massa superiore (Figura 9C), e così pure SALL4, un repressore trascrizionale che ha anche dimostrato di impegnare nurd 20, 24, 25. Così, frazionamento di affinità purificate proteine ​​MTA2-associata di blu PAGE nativa è riuscito a risolvere due diverse forme del complesso nurd. ABC-MS permette anche l&#39;identificazione di nuovi interattori mentre assegnandoli a particolari entità proteiche. Attraverso l&#39;esame dei cluster proteine e intensità frazione rappresentata in una mappa termica abbiamo rilevato una forte correlazione tra subunità nurd e Wdr5, una subunità regolatrice del complesso metiltransferasi MLL 26, con Wdr5 visualizzazione due picchi migrazione coincidenti con i due nurd peaks (Figura 8), suggerendo una nuova interazione tra Wdr5 e nurd. Abbiamo confermato questa interazione mediante co-immunoprecipitazione e co-migrazione cromatografia di esclusione dimensionale 20. La scelta di calcolo della metrica distanza nel clustering gerarchico influenzerà la forma dei cluster e quindi le correlazioni. Si consiglia di sperimentare le diverse metriche per raggiungere la migliore forma con la conoscenza dell&#39;interazione esistente della proteina o complessi di interesse. Generalmente abbiamo ottenuto risultati migliori con il Manhattan (L1) distanza metrica 20. Correlazione di Pearson o metriche distanza euclidea sono stati riportati anche per identificare complessi basati su tecniche di frazionamento alternative 10, 11, 12. la quandati titative MS ottenuti da frazioni native blu possono anche essere utilizzati per determinare la stechiometria dei complessi proteici. Il valore iBAQ (intensità basato assoluta quantificazione) fornisce una misura della relativa abbondanza di proteine identificate 27, 28. I valori iBAQ per la proteina membri complessi in tutte le frazioni all&#39;interno di un profilo di migrazione picco vengono normalizzati a quella della proteina esca per ottenere quantità relative. I iBAQs normalizzati attraverso un profilo di picco di un membro complessa data proteina dovrebbero seguire un andamento orizzontale, e i valori delle curve riflettere la stechiometria delle proteine ​​interagenti relativi alla proteina esca. Per una rappresentazione più dettagliata di calcoli stechiometrici, vedi 20. <img alt="Figura 7" src="/files/ftp_upload/55498/55498fig7.jpg" /> Figura 7: Schema flusso che sintetizza l&#39;ABC-MS sSTRATEGIA. Questa figura è modificata da 20. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55498/55498fig7large.jpg" target="_blank">Si prega di cliccare qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 8" src="/files/ftp_upload/55498/55498fig8.jpg" /> Figura 8. raggruppamento gerarchico dei profili migratori BN-PAGE di interattori MTA2. proteine ​​MTA2-associati sono stati raggruppati in base alla somiglianza delle loro profili migratori. È mostrata solo un sottoinsieme della mappa termica contenente il complesso nurd (racchiuso nella scatola blu). La scatola gialla evidenzia la forte correlazione del Wdr5 con il complesso nurd. I pesi molecolari annotati sono stati stimati dalle distanze di migrazione degli standard proteine ​​eseguite sullo stesso gel. Questa ricerca è stato originariamente pubblicato in molecolare e cellulare Proteomica 20 l&#39;American Society for Biochimica e Mo<html> Biologia lecular. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55498/55498fig8large.jpg" target="_blank">Si prega di cliccare qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 9" src="/files/ftp_upload/55498/55498fig9.jpg" /> Figura 9. BN-PAGE profilo migrazione delle subunità nurd e proteine associate. A) subunità MTA2 e nurd presenti in un modello simile intensità. B) MTA2 e nurd subunità con il modello intensità inversa. C) MTA2 e Cdk2ap1, che è presente solo nell&#39;entità nurd peso molecolare superiore. I pesi molecolari annotati sono stati stimati dal profilo migrazione degli standard proteina. Questa ricerca è stato originariamente pubblicato in Cellular Proteomics 20 l&#39;American Society for Biochimica e Biologia Molecolare Molecolare e._blank &quot;&gt; Clicca qui per vedere una versione più grande di questa figura. <img alt="File supplementare" src="/files/ftp_upload/55498/proteinGroupsDB04.jpg" /> Dati supplementari: set di dati campione. MaxQuant-derivato del file contenente proteingroups.txt identificazioni di proteine ​​e valori di quantificazione da un esperimento ABC-MS. Questa ricerca è stato originariamente pubblicato in Cellular Proteomics 20 l&#39;American Society for Biochimica e Biologia Molecolare Molecolare e. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55498/proteinGroupsDB04.txt" target="_blank">Cliccate qui per scaricare questo file.</a>

Watch this Scientific Journal Video about Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling at JoVE.com

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Presentiamo qui protocolli per purificazione per affinità di complessi proteici e la loro separazione dal blu PAGE nativa, seguiti da proteine ​​correlazione profilatura mediante un&#39;etichetta disponibile spettrometria di massa quantitativa. Questo metodo è utile per risolvere interattomi in distinti complessi proteici.

Risoluzione purificato per affinità complessi proteici di Blue Native PAGE e proteine ​​correlazione Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

resolving-affinity-purified-protein-complexes-blue-native-page

Nanyang Technological University

Research

JoVE Journal

Biochemistry

13.7K Views.  Wellcome Trust Sanger Institute.  Presentiamo qui protocolli per purificazione per affinità di complessi proteici e la loro separazione dal blu PAGE nativa, seguiti da proteine ​​correlazione profilatura mediante un'etichetta disponibile spettrometria di massa quantitativa. Questo metodo è utile per risolvere interattomi in distinti complessi proteici. 

Video: Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Proteine ​​Complesso affinità Acquisizione da cellule di mammifero Cryomilled

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

Ricerca

Biochimica

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

Tandem Affinity Purification di complessi proteici da cellule eucariotiche

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by networks of interacting protein complexes, which are often difficult to investigate because their binding is non-covalent and easily perturbed by purification techniques. This work describes a method of stabilizing and separating native protein complexes from unmodified tissue using two-dimensional polyacrylamide gel electrophoresis. Tissue lysate is loaded onto a non-denaturing blue-native polyacrylamide gel, then an electric current is applied until the protein migrates a short distance into the gel. The gel strip containing the migrated protein is then excised and incubated with the amine-reactive cross-linking reagent dithiobis(succinimidyl propionate), which covalently stabilizes protein complexes. The gel strip containing cross-linked complexes is then cast into a sodium dodecyl sulfate polyacrylamide gel, and the complexes are separated completely. The method relies on techniques and materials familiar to most molecular biologists, meaning it is inexpensive and easy to learn. While it is limited in its ability to adequately separate extremely large complexes, and has not been universally successful, the method was able to capture a wide variety of well-studied complexes, and is likely applicable to many systems of interest.

A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented.

Multimer-PAGE: Un metodo per acquisire e risolvere i complessi proteici nei campioni biologici

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by ...

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of RNA-protein interactions. These interactions have been traditionally analyzed with polyacrylamide gels generated between two glass plates and samples electrophoresed vertically. However, polyacrylamide gels cast in trays and electrophoresed horizontally offers several advantages. For example, horizontal gels used to analyze complexes between fluorescent RNA substrates and specific proteins can be imaged multiple times as electrophoresis progresses. This provides the unique opportunity to monitor RNA-protein complexes at several points during the experiment. In addition, horizontal gel electrophoresis makes it possible to analyze many samples in parallel. This can greatly facilitate time course experiments as well as analyzing multiple reactions simultaneously to compare different components and conditions. Here we provide a detailed protocol for generating and using horizontal native gel electrophoresis for analyzing RNA-Protein interactions.

Native polyacrylamide gel electrophoresis is a fundamental tool for analyzing RNA-protein interactions. Traditionally most experiments have used vertical gels. However, horizontal gels provide several advantages, such as the opportunity to monitor complexes during electrophoresis. We provide a detailed protocol for generating and using horizontal native gel electrophoresis.

Elettroforesi gel orizzontale per la rilevazione avanzata dei complessi proteina-RNA

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of ...

Split-BioID &#8212; Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced that allow the proximity-dependent labeling of proteins in living cells. One such enzyme, BirA* (used in the BioID approach), mediates the biotinylation of proteins within a range of approximately 10 nm. Hence, when fused to a protein of interest and expressed in cells, it allows the labeling of proximal proteins in their native environment. As opposed to AP that relies on the purification of assembled protein complexes, BioID detects proteins that have been marked within cells no matter whether they are still interacting with the protein of interest when they are isolated. Since it biotinylates proximal proteins, one can moreover capitalize on the exceptional affinity of streptavidin for biotin to very efficiently isolate them. While BioID performs better than AP for identifying transient or weak interactions, both AP- and BioID-mass spectrometry approaches provide an overview of all possible interactions a given protein may have. However, they do not provide information on the context of each identified PPI. Indeed, most proteins are typically part of several complexes, corresponding to distinct maturation steps or different functional units. To address this common limitation of both methods, we have engineered a protein-fragments complementation assay based on the BirA* enzyme. In this assay, two inactive fragments of BirA* can reassemble into an active enzyme when brought in close proximity by two interacting proteins to which they are fused. The resulting split-BioID assay thus allows the labeling of proteins that assemble around a pair of interacting proteins. Provided these two only interact in a given context, split-BioID then allows the analysis of specific context-dependent functional units in their native cellular environment. Here, we provide a step-by-step protocol to test and apply split-BioID to a pair of interacting proteins.

We provide a step-by-step protocol for split-BioID, a protein fragments-complementation assay based on the proximity-labeling technique BioID. Activated on the interaction of two given proteins, it allows the proteomics analysis of context-dependent protein complexes in their native cellular environment. The method is simple, cost-effective and only requires standard laboratory equipment.

Split-BioID — Analisi proteomica dei complessi della proteina contesto specifico in ambiente nativo di cellulare

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2 in 1: purificazione di affinità per l'analisi parallela di proteina-proteina e proteina-metabolita complessi in un unico passaggio

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

Analisi dei complessi della proteina di membrana tilacoide mediante elettroforesi nativa blu

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.

Purificazione di affinità del cloroplasto Translocon proteina complessi usando il Tag di rubinetto

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

Alta sensibilità misura dell'affinità di legame di DNA di fattore di trascrizione per titolazione competitivo mediante microscopia a fluorescenza

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Analisi della piegatura della proteina, trasporto e degradazione in cellule viventi di radioattivo Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Risoluzione purificato per affinità complessi proteici di Blue Native PAGE e proteine ​​correlazione Profiling

Risoluzione purificato per affinità complessi proteici di Blue Native PAGE e proteine correlazione Profiling