Questo lavoro è stato sostenuto dalle concessioni dal National Institutes of Health a VF (NS42599 e GM077569).

1. Preparazione per reticolazione <ol><li> Avrete bisogno di piatto un numero sufficiente di celle per consentire l&#39;isolamento di 500 mg di proteine ​​per reazione tubo standard. Un tubo singolo può essere sufficiente per l&#39;identificazione di putativi interattori di una proteina di interesse da immunoblot. In questo caso tallone materiale legato può essere eluito con SDS-PAGE tampone del campione (immunoprecipitazione). Per l&#39;analisi di spettrometria di massa, il numero di reazioni standard dovrebbe essere aumentata almeno dieci volte e complessi di proteine ​​deve essere eluita da fuori concorso con un antigene peptide riconosciuto dal anticorpo utilizzato (immunoaffinità cromatografia). Questa strategia permetterà l&#39;isolamento di complessi proteici liberi di immunoglobuline. </li><li> La densità cellulare ottimale per reticolazione è compresa tra 75% e il 90% confluenza. In condizioni di elevata confluenza, le cellule tendono ad accumularsi uno sull&#39;altro, che diminuisce l&#39;accessibilità alla cella-permeabile reticolante a tutte le celle. </li><li> Tipo di cellula e dalle condizioni sperimentali devono essere etichettati sul fondo della piastra cellulare. Ci sono controlli di routine con solo veicolo. </li><li> Preparare tutte le soluzioni TRANNE la soluzione DSP prima di reticolazione. <ol><li> Preparare uno stock di tampone fosfato tampone salino con CaCl 2 0,1 mM e CaCl 2 1 mM (PBS / Ca / Mg) prima di iniziare l&#39;esperimento. L&#39;aggiunta di ioni calcio e magnesio è fondamentale per l&#39;adesione delle cellule alla piastra di coltura nel corso dell&#39;esperimento. Conservare a 4 ° C. </li><li> 50X completa inibitori della proteasi Cocktail - 1 compressa sciolta in 1 ml di acqua Milli-Q. Conservare a -20 ° C. </li><li> 20% di Triton X-100 Pesare 10g Triton X -100 e diluire a volume totale di 50 ml di acqua Milli-Q. Roccia a 4 ° C per una notte e conservare a 4 ° C fino all&#39;uso. Non conservare per più di 1 mese. Utilizzare diluizioni di questo stock del 20% per preparare il buffer di lisi e IP descritto di seguito. </li><li> 50X DSP tempra Soluzione TRIS 1M a pH 7.4. Conservare a temperatura ambiente. </li><li> Preparare un tampone 10x Una soluzione. Buffer 10X A: <ul><li> 50 ml 1 M HEPES </li><li> 150 ml di NaCl 5 M </li><li> 10 mL 0,5 M EGTA </li><li> 250 microlitri 2 M MgCl 2 </li><li> Regolare il pH a 7,4 </li><li> Portare il volume finale a 500 ml </li></ul> Il tampone 10X Una soluzione viene diluita a 1X tampone A seconda delle necessità. Buffer 1x A è utilizzato anche in lisato e immuno-magnetico-precipitazione buffer. Un tampone 1X <ul><li> 10 mM Hepes </li><li> 150 mM NaCl </li><li> 1 mM EGTA </li><li> 0.1 mM MgCl 2 </li><li> pH 7,4 </li></ul></li><li> Tampone di Lysis Buffer 1X A + 0,5% Triton X-100. </li><li> Immuno-magnetica Buffer Precipitazioni (IP Buffer) 1X tampone A + 0,1% Triton X-100. </li></ol></li></ol> 2. Preparazione della soluzione di reticolazione <ol><li> Preparare la soluzione DSP immediatamente prima di applicare alle cellule. DSP è altamente idrofoba e deve essere sciolto in DMSO prima di diluire in PBS / Ca / Mg buffer. Sciogliere 40 mg di DSP in 1 ml di DMSO. Questo rende una soluzione 100 mM di DSP (Il peso molecolare di DSP è 404,42 g / mol). </li><li> Caldo un volume adeguato di PBS / Ca / Mg a 37 ° C al fine di facilitare la diluizione del DSP / DMSO in PBS. Volumi necessari per dimensioni piatto assortito: 6-pozzetti 2 mL per pozzetto 10 centimetri tavola 10 mL per piastra 15 centimetri piatto 20 ml per piastra </li><li> Aggiungere 10μL di DSP / DMSO soluzione madre per ogni 1 ml di PBS caldo / Ca / Mg. Aggiungi DSP / DMSO goccia a goccia soluzione madre con miscelazione ripetuto fino a quando tutti i DSP si è dissolto. Ottenere una soluzione di controllo di 10 microlitri di DMSO aggiunto ad ogni 1 ml di PBS / Ca / Mg. </li><li> Mettere tutti PBS / Ca / Mg soluzioni in un bagno di acqua e ghiaccio non più di 10 min. </li></ol> 3. Preparare Celle per reticolazione <ol><li> Preparare un bagno di ghiaccio d&#39;acqua che si adatta tutte le lastre di reticolazione o controllo di incubazione del veicolo. </li><li> Prendere le piastre dal 37 ° C incubatore e metterli immediatamente in ghiaccio acqua sporca. </li><li> Lavare le cellule due volte con gelida PBS / Ca / Mg. Utilizzare lo stesso volume che verrà utilizzato per l&#39;incubazione reticolante (vedi sezione 2.2). </li><li> Rimuovere il secondo lavaggio e aggiungere il controllo di veicoli o di una soluzione tampone reticolazione DSP. </li><li> Incubare in ghiaccio per due ore. Si dovrebbe controllare le piastre circa ogni venti minuti per assicurare che tutte le cellule sono coperti da una soluzione. Si può notare una piccola quantità di DSP precipitare dalla soluzione. Questo è normale. </li></ol> 4. Inattivazione di reazione DSP <ol><li> Preparare una soluzione 1X tempra DSP di 20 mM Tris pH 7.4 in PBS / Ca / Mg (20 ml di 1 M Tris pH 7,4 per ogni 1 ml di PBS / Ca / Mg). </li><li> Rimuovere il controllo del veicolo e le soluzioni reticolazione. </li><li> Aggiungi ghiacciata soluzione inattivazione e incubare in ghiaccio per 15 minuti. </li></ol> 5. Cellula Lysis <ol><li> Durante l&#39;incubazione tempra DSP, preparare il tampone di lisi delle cellule del 0,5% Triton X-100 in tampone A con cocktail Completa inibitore della proteasi. Aggiungere 20 ul di soluzione di 50X magazzino completo per ogni buffer 1 lisi ml. </li><li> Seguendo i 15 minuti di incubazione tempra DSP, lavare le cellule due volte con PBS / Ca / Mg. </li><li> Aggiungi tampone di lisi + completa alle cellule e rock a 4 ° C per 30 minuti. Alcuni hanno suggerito volumi di tampone di lisi per dimensioni piatto assortito: <ul><li> 6-pozzetti 0,5 mL per pozzetto </li><li> 10 centimetri piastra 1 ml per piastra </li><li> 15 centimetri piastra 1 ml per piastra </li></ul></li><li> Dopo la lisi, usare un raschietto cellula per raccogliere le cellule dalla piastra. Essere sicuri di mantenere lisati sul ghiaccio per ridurre al minimo l&#39;attività della proteasi </li><li> Spin lisati cellulari per 15 minuti a 4 ° C in un banco mini-centrifuga alla massima velocità (15.000 xg). </li><li> Pipettare il supernatante in una nuova provetta. Eliminare il pellet. </li><li> Analizzare i livelli di proteine ​​e procedere con immunoprecipitazione. </li></ol> 6. Preparazione Immuno-magnetico Perline Precipitazioni Nota: questo passaggio è di solito avviato direttamente dopo l&#39;inizio l&#39;incubazione reticolazione 2 ore. <ol><li> Unire 30 ml di Dynal sfere immunomagnetiche, 500 ml di IP Buffer (1x tampone A + 0,1% Triton X-100) e la giusta quantità di antigene-anticorpo specifico per screwtop tubi microcentrifuga. Preparare anticorpi liberi e non specifici anticorpi tubi per i controlli negativi. La giusta quantità di anticorpi deve essere determinato sulla base di singoli antigeni durante gli esperimenti separati. Etichetta tutti i tubi IP. </li><li> Lasciare i tubi IP ad incubare in una soluzione end-over-end rocker a temperatura ambiente per due ore. In alternativa, incubare i tubi IP con anticorpi overnight a 4 ° il giorno prima della reticolazione. </li><li> Dopo l&#39;incubazione, fare un rapido 10 secondi mini-centrifugazione per raccogliere le perle sul fondo della provetta. </li><li> Far scorrere i tubi nel supporto magnetico, che farà rientrare il sfere magnetiche lontano dal fondo della provetta. Con le perle in modo sicuro fuori del modo, si può ora facilmente lavare via ogni anticorpo non legato. </li><li> Utilizzare una piccola mancia aspiratore portava come una punta di caricamento del gel, o P200 punta a rimuovere il tampone di incubazione e qualsiasi anticorpo non legato. Non appena il volume di incubazione è stato rimosso aggiungere in 1 ml di IP Buffer. </li><li> Chiudere la provetta, delicatamente risospendere le perline, e tutti i tubi IP incubare a temperatura ambiente per 5 minuti con end-over-end di rotazione. </li><li> Ripetere la fase di lavaggio 5 minuti (6,3 punti con 6,6) ancora una volta con un millilitro fresco di IP Buffer. </li><li> I tubi IP può rimanere in incubazione ultimo lavaggio fino a quando il lisato reticolato è pronto per essere aggiunto ai talloni. </li></ol> 7. Incubare lisato reticolato con Immuno-magnetico Perline <ol><li> Dopo l&#39;ultimo lavaggio di spin immuno-magnetico perline, giù in una mini-centrifuga per 10 secondi. Quindi procedere a scivolare i tubi nel supporto magnetico. </li><li> Ancora una volta, con una piccola mancia aspirazione foro, rimuovere il volume di incubazione dal tubo. Immediatamente aggiungere 500 microlitri di lisato cellulare reticolato (assumendo lisato a 1 mg / ml) per i tubi contenenti immuno-magnetico perline. Se si prevede di utilizzare concorrenza peptide come controllo negativo, una quantità appropriata di peptide dovrebbe essere incluso, a questo punto. Precedenti esperimenti devono essere effettuati per determinare adeguate condizioni di concorrenza peptide. In alternativa, si consiglia di avere lisato libero immuno-magnetico perle come controllo negativo. In questo caso, immediatamente aggiungere 500 ml di lisato Buffer (1x tampone A + 0,5% Triton X-100) + completa per immuno-magnetico perline. </li><li> Risospendere delicatamente le sfere. Microsfere in sospensione devono essere incubate a 4 ° in un fine-over-end di rotazione per 2 ore. </li></ol> 8. Lavare LISATO Unbound di Perline <ol><li> Una volta lisato ha avuto il tempo sufficiente incubazione, fare un rapido giro 10 secondi in un mini-centrifuga. Quindi, far scorrere i tubi nel supporto magnetico. Il supporto magnetico deve essere conservato in un bagno di ghiaccio per il resto l&#39;esperimento. </li><li> Utilizzando una piccola mancia aspiratore foro, rimuovere tutti i lisato non legato. Immediatamente dopo che tutti lisato non legato viene rimosso aggiungere 1 ml di IP Buffer. </li><li> Chiudere la provetta e delicatamente risospendere le sfere. </li><li> Una volta che le perle sono risospeso ripetere il passaggio tallone pellettizzazione (8,1). </li><li> Ancora una volta aspirato fuori tutto il volume di incubazione e subito aggiungere 1 ml di IP Buffer, poi tappo il tubo. </li><li> Risospendere delicatamente le perle in IP Buffer. Incubare microsfere in sospensione per 5 minuti a 4 ° con end-over-end a dondolo. </li><li> Ripetere le operazioni di lavaggio tallone (8,4 per 8,6) 4 volte di più. </li></ol> 9. Denaturare campione e raccogliere da perline <ol><li> Dopo che tutte le immuno-magnetico tubi precipitazione sono stati lavati, ri-sedimento le perline (8,1) e tubi di scivolare nellasupporto magnetico. </li><li> Le proteine ​​legate alla immuno-magnetico perline vengono rimossi in condizioni di denaturazione. Aspirare fuori l&#39;ultimo lavare IP Buffer e rimuovere il tubo IP dal supporto magnetico. </li><li> Aggiungi 1x Sample Buffer Gel al tubo IP appena sopra il pellet tallone mettere in comune le perline sul fondo del tubo di nuovo. La quantità di tampone campione dipende dalle dimensioni e del gel da utilizzare. Potrebbe essere necessario premere delicatamente il fondo della provetta per ottenere tutte le microsfere in sospensione nel Sample Buffer Gel. </li><li> Ripetere il processo di denaturazione (9,2 per 9,3) per ogni tubo IP. </li><li> Immuno-magnetico perline risospese nel tampone Gel campioni vengono poi riscaldate a 75 ° C per 5 minuti per completare il processo di denaturazione. </li><li> Una volta che il campione è stato denaturato può essere eseguito su un gel SDS-PAGE per blotting occidentale. Svuotato Immuno-Magnetic Beads può essere ri-pellet per centrifugazione e rimosso dal campione con il supporto magnetico. </li></ol> 10. Eluizione dei complessi reticolato con peptidi antigenici (immunoaffinità Chromatography). <ol><li> Dopo che tutte le immuno-magnetico tubi immunoprecipitazione sono stati lavati, ri-sedimento le perline (8,1) e tubi diapositiva nel supporto magnetico. Aspirare il surnatante e aggiungere 10 ml di tampone A integrato con il peptide antigenico riconosciuto dagli anticorpi utilizzati per immunoisolamento di complessi proteici. È necessario verificare concentrazioni comprese 1-20 mM per l&#39;eluizione efficiente. </li><li> Incubare le perline per 2 ore a 4 ° C. </li><li> Mettere perle nello stand magnetico e con attenzione recuperare i 10 ml di materiale eluito. Salva questo supernatante. </li><li> Lavare rapidamente le perline con tampone A e scartare il lavaggio. Salva le perline. </li><li> Aggiungi Sample buffer Gel per il supernatante salvato e perline per una concentrazione di 1X. Incubare le provette a 75 ° C per 5 minuti. </li><li> Analizzare perline e eluato peptide mediante SDS-PAGE. L&#39;eluato dovrebbe essere libero di IgG sia da proteine ​​macchia di SDS-PAGE o immunoblot (Figura 1B). Questo materiale libero di IgG è adatto per spettrometria di massa. </li></ol><img src="/files/ftp_upload/1855/1855fig1.jpg" alt="Figura 1" /> Figura 1. Isolamento di AP-3 complessi di proteine ​​che interagiscono e proteine ​​di membrana. Cellule HEK293 (A) o cellule PC12 (B) sono stati trattati sia in presenza di controllo del veicolo (DMSO, dispari corsie Fig. 1A) o DSP (anche corsie fig. 1A o tutti corsie in Fig. 1B). Estratti chiarito sono state incubate sia con perline da solo (Fig. 1A, Lanes 3-4), anticorpi contro il recettore della transferrina (Fig. 1A, Lanes 5-6), o AP-3 anticorpi δ (Fig. 1A, Lanes 7-10; Fig. . 1B, corsie 1-10). Immunocomplessi sono stati eluiti con SDS-PAGE tampone del campione (Fig. 1A), tampone A soli o (Fig. 1B, corsie 1-2), o tampone A integrato con concentrazioni crescenti del peptide antigenico δ corrispondente al aminoacidi 680 710 delle risorse umane δ-adaptin (NCBI: AAD03777; gi: 1923266) (Fig. 1B, corsie 3-10). Questo peptide si lega l&#39;anticorpo δ. Dopo l&#39;eluizione peptide, surnatanti (S) e perline (B) sono stati analizzati mediante immunoblot (Fig. 1B). AP-3, che viene rilevata con anticorpi contro il δ, β3 e σ3 subunità, coprecipitates con i seguenti fattori solubili: clatrina catena pesante (CHC), il BLOC-1 pallidin subunità, e il luppolo vps33b subunità complesso, così come le proteine ​​di membrana fosfatidil-4-chinasi di tipo II alfa (PI4KIIα) e il trasportatore di zinco 3 (ZnT3). Si noti l&#39;assenza di IgG catene del mouse pesanti nel peptidi eluiti surnatante in fig. 1B.

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

DSP, una membrana permeabile, reticolante chimicamente riducibili con un braccio distanziatore di 12 A viene utilizzato per stabilizzare le interazioni proteina transitoria 1,2,3,4. Qui abbiamo esemplificato questa strategia con il complesso adattatore AP-3 un complesso proteina solubile che riconosce e ordina proteine ​​di membrana in vescicole da endosomi 5. AP-3 si lega selettivamente al trasportatore di zinco ZnT3 e la chinasi dei lipidi fosfatidilinositolo-4-chinasi di tipo II, ma non dei ...

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

La natura dinamica di macchine cellulare è spesso costruito sulle associazioni di proteine ​​transitori e / o debole. Queste interazioni bassa affinità preclude metodi rigorosi per l&#39;isolamento e l&#39;identificazione di reti di proteine ​​intorno ad una proteina di interesse. L&#39;uso di reticolanti chimici permette la stabilizzazione selettiva delle interazioni labili, aggirando così le limitazioni biochimiche per la purificazione. Qui vi presentiamo un protocollo suscettibile di cellule in coltura che utilizza un reticolante homobifunctional con un braccio distanziatore di 12 Å, dithiobis-(succinimidile propionato) (DSP). DSP è scissa dalla riduzione di un legame disolfuro presenti nella molecola. Cross-linking in combinazione con la cromatografia di immunoaffinità di proteine ​​di interesse con perline magnetiche permette l&#39;isolamento di complessi proteici che altrimenti non avrebbe resistere purificazione. Questo protocollo è compatibile con le normali tecniche di Western Blot e può essere scalata per l&#39;identificazione delle proteine ​​mediante spettrometria di massa 1. Stephanie A. Zlatic e Pearl V. Ryder contribuito in maniera uguale a questo lavoro.

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

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

La cellula permeabile reticolante DSP [dithiobis-(succinimidile propionato)] stabilizza le interazioni transienti e labile<em&gt; In vivo</em&gt;, Che consente loro isolamento utilizzando rigorosi tecniche di purificazione di proteine ​​complesse. Qui vi presentiamo una tecnica per la reticolazione cellule in coltura seguita da isolamento di complessi proteici da immunoprecipitazione.

Isolamento di Labile Multi-proteina complessi da In vivo Controllato Cellular Cross-Linking e Immuno-magnetica Cromatografia di affinità

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

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Biology

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

17.2K Views.  Emory University. La cellula permeabile reticolante DSP [dithiobis-(succinimidile propionato)] stabilizza le interazioni transienti e labile In vivo, Che consente loro isolamento utilizzando rigorosi tecniche di purificazione di proteine ​​complesse. Qui vi presentiamo una tecnica per la reticolazione cellule in coltura seguita da isolamento di complessi proteici da immunoprecipitazione.

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

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

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

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

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

Photo-Induced Cross-Linking delle proteine ​​non modificati (PICUP) applicata a peptidi amiloidogenico

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

Ricerca

Biologia

Identification of protein complexes with quantitative proteomics in S. cerevisiae

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

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

L&#39;identificazione di complessi proteici con la proteomica quantitativa in S. cerevisiae

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

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

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

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

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

Visualizzazione singola complessi molecolari In Vivo Utilizzo avanzato microscopia a fluorescenza

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

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

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

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

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

ELIME (Enzyme Linked Immuno magnetica elettrochimici) metodo di rilevazione micotossine

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

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

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

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

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

Imaging in vivo di intatto Larve di Drosophila a livello sub-cellulare Risoluzione

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

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

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

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

Calorimetria isotermica di titolazione di misura macromolecola-ligando di affinità

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

Chromatin Isolation by RNA Purification (ChIRP)

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

Chromatin Isolation by RNA Purification ChIRP

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

Isolamento di cromatina Purificazione RNA (disturbo)

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

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

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

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

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

Identificazione di complessi proteici in<em&gt; Escherichia coli</em&gt; Con sequenziale purificazione per affinità Peptide in combinazione con Tandem Mass Spectrometry

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

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

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

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

Mapping basato AFM delle proprietà elastiche delle pareti cellulari: al Tissue, cellulare, e le risoluzioni subcellulari

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

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

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

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

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

In Vivo inseguimento 4-dimensionale di emopoietiche staminali e progenitrici celle di topo adulto cranica di Midollo Osseo

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