Questo lavoro è stato sostenuto dal Ministero dell&#39;Economia e della Compagnia / Instituto de Salud Carlos III (SAF2014-55700-P, PCIN-2013-019-C03-03 e PIE14 / 00034), Institutional Catalonia de Recerca i Estudis Avançats (ICREA Academia-2010 (SBO-140028) a FC Inoltre, X. M, VF-D e FC appartengono al gruppo di ricerca accreditato &quot;Neuropharmacology and Pain&quot; (Generalitat de Catalunya, 2014 SGR 1251) . Il lavoro è stato sostenuto anche dal &quot;Programa Pesquisador Visite Especial-Ciência sem Fronteiras&quot; da CAPES (Brasile) a FC

Tutte le procedure sperimentali animali sono state approvate dal Comitato di Barcellona per l&#39;Uso e la Cura degli Animali (CEEA), conformemente alle linee guida descritte nella Guida per la cura e l&#39;uso degli animali da laboratorio11 e seguendo la Comunità Europea, la legge 86/609 / CCE, FELASA e ARRIVE. Così, i topi sono alloggiati in gabbie standard, con accesso ad libitum all&#39;alimentazione e all&#39;acqua e vengono mantenute in condizioni standard controllate (ciclo scuro / leggero da 12 ore a partire da 7:30 AM, temperatura di 22 ° C e umidità del 66%). 1. Ottenere Synaptosomes del cervello del mouse usando un Gradiente Discontinuo di Sucroso Nota: Questo metodo è stato segnalato in precedenza 10 . <ol><li> Preparare tampone di isolamento fresco (IB), pH 7,4; 2 M saccarosio; 1 M saccarosio / 0,1 mM CaCl2; E 0,1 mM CaCl2 (vedi tabella 1 ). Chill le soluzioni sul ghiaccio. </li><li> SaCrizione di cinque topi per dislocazione cervicale. Decapitate e rimuovete rapidamente il cervello di ogni animale. Disciogliere la regione cerebrale di interesse (cioè l&#39;ippocampo, 0,25-0,35 g di tessuto fresco) e metterlo in un tubo di vetro Potter-Elvehjem da 5 ml su ghiaccio con 1 ml di IB. </li><li> Homogenizzare il tessuto utilizzando un agitatore omogeneizzante (10 colpi a 700-900 rotazioni al minuto), preferibilmente in un bagno di ghiaccio per evitare il riscaldamento del campione. </li><li> Posizionare il tessuto omogeneizzato (1 mL) in un tubo da 15 mL contenente 6 mi di saccarosio da 2 M e 2,5 mL di 0,1 mM CaCl2 a 4 ° C. Mescolare lentamente invertendo il tubo. Nota: l&#39;aggiunta di calcio è essenziale per mantenere le interazioni adesive tra gli organelli e quindi per preservare la struttura delle diverse &quot;densità&quot;. </li><li> Posizionare la soluzione in un tubo da centrifuga da 12 ml e aggiungere lentamente 2,5 ml di saccarosio di 1 M / 0,1 mM CaCl2 sopra ogni tubo per formare il gradiente di saccarosio. Nota: contrassegnare la posizione oF l&#39;interfaccia di gradiente sul tubo centrifuga utilizzando un indicatore permanente. </li><li> Pesare e equilibrare i tubi di centrifuga con soluzione IB all&#39;interno dei rispettivi supporti in acciaio e con i loro coperchi di chiusura. </li><li> Centrifugare i tubi per 3 ore a 100.000 x g e 4 ° C utilizzando una centrifuga a rotore a pozzo. Riempire completamente il rotore con tutti i supporti in acciaio (anche vuoto, se necessario). </li><li> Scartare il livello superiore contenente mielina. Con una pipetta Pasteur raccogliere l&#39;anello bianco tra l&#39;interfaccia di saccarosio 1,25-M e 1-M corrispondente alla frazione sinaptosoma. </li><li> Diluire i sinaptosomi con 9X il loro volume usando IB in un tubo di centrifuga. </li><li> Pesare e equilibrare i tubi di centrifuga con soluzione IB all&#39;interno dei rispettivi supporti in acciaio e con i loro coperchi di chiusura. </li><li> Centrifugare i tubi per 30 minuti a 15.000 x g e 4 ° C utilizzando una centrifuga a rotore a pozzo. </li><li> Scartare il surnatante e riposizionareE con 10 ml di soluzione IB. Raccogliere 100 μL di questa soluzione sinaptosomica e centrifugare per 5 minuti a 11.000 x g e 4 ºC. </li><li> Riposizionare il pellet sinaptosomico in sodio dodecil solfato (SDS) al 5% e memorizzare questo campione a -20 ° C. Nota: Questo campione corrisponderà alla frazione totale di sinaptosomi per ulteriori analisi Western blot. La restante frazione sinaptosomica (~ 1 ml) può essere congelata a -20 ° C fino all&#39;uso o elaborata per la successiva frazionamento subsintattico. I sinaptosomi o le sinapsi purificati rappresentano tra il 1 e il 2% del volume totale ippocampale 12 . </li></ol> 2. Isolamento pre-, post- ed extrasinaptico <ol><li> Preparare un tampone di solubilizzazione fresco 2x, pH 6,0; 1x tampone di solubilizzazione, pH 6,0; Tampone di solubilizzazione, pH 8,0; E 0,1 mM CaCl2 (vedi tabella 1 ). Chill le soluzioni sul ghiaccio. Nota: il pH di queste soluzioni dovrebbe essere accuratelY regolato per ottenere una buona frazionamento subsynaptic. </li><li> Diluire lentamente la frazione sinaptosomale resuspendente da 1 mL (vedere la fase 1.12) con 5 mL di 0,1 mM CaCl2. </li><li> Aggiungere lo stesso volume (5 mL) di tampone di solubilizzazione 2x a ghiaccio freddo, pH 6,0 e incubare per 50 minuti su ghiaccio sotto agitazione elevata in un bicchiere su ghiaccio. Nota: mentre l&#39;1% di Triton X-100 a pH 6.0 solubilizza tutte le proteine ​​della membrana plasmatica, conserva le proteine ​​all&#39;interno dei contatti o dei giunti sinaptici ( vale a dire, strutture pre e postsynaptiche). </li><li> Mettere la soluzione in un tubo di centrifuga. Pesare e equilibrare i tubi con volumi equivalenti di 0,1 mM CaCl2 e 2x soluzione tampone di solubilizzazione, pH 6,0, all&#39;interno dei rispettivi supporti in acciaio e con i loro coperchi di chiusura. </li><li> Centrifugare i tubi per 30 minuti a 40.000 x g e 4 ° C utilizzando una centrifuga a rotore a pozzo. Il supernatante risultante rappresenta la frazione extrasinaptica,E il pellet corrisponde alle giunzioni sinaptiche ( cioè, frazioni pre- e postinaptiche). </li><li> Concentrare il supernatante contenente la frazione extrasinaptica a un volume finale di 200 μL usando un tubo filtrante da 15 mL di 10K centrifugato a 4.000 xg e 4 ºC. </li><li> Precipitare la frazione extrasinaptica risultante risultante (200 μL) con 5 volumi (1 ml) di acetone pre-refrigerato (-20 ° C) per una notte a -20 ° C. </li><li> Il giorno dopo, centrifuga la frazione extrasinaptica per 30 minuti a 18.000 x g e -15 ° C. Dopo la centrifugazione, scartare il supernatante di acetone e asciugare il pellet per rimuovere qualsiasi traccia di acetone. </li><li> Infine, risospendere il pellet contenente le proteine ​​extrasinaptiche con 200 μL di 5% SDS. Sonicare il pellet, se necessario. </li><li> Senza interromperla, lavare attentamente il pellet contenente le frazioni pre- e postsinaptiche con 2 mL di tampone di solubilizzazione 1x, pH 6,0. Scartare il buffer. </li><li> RisospendereIl pellet con 10 ml di tampone di solubilizzazione 1x, pH 8,0, utilizzando una pipetta di vetro Pasteur. Nota: mentre l&#39;1% di Triton X-100 a pH 8,0 solubilizza la specializzazione presinaptica, conserva la densità postinaptica insolubile 4 . </li><li> Incubare la sospensione per 50 minuti su ghiaccio sotto agitazione elevata in un bicchiere su ghiaccio. </li><li> Mettere la soluzione in un tubo di centrifuga. Pesare e equilibrare i tubi con una soluzione di tampone di soluzione solubilizzazione, pH 8,0, all&#39;interno dei relativi supporti in acciaio e con i loro coperchi di chiusura. </li><li> Centrifugare i tubi per 30 minuti a 40.000 x g e 4 ° C utilizzando una centrifuga a rotore a cilindro oscillante; Il supernatante risultante corrisponde alla frazione presinaptica e al pellet alla frazione postsinaptica. </li><li> Processare il surnatante contenente la frazione presinaptica, come descritto nei passaggi 2.6-2.9. </li><li> Resuspendere la pellicola contenente la frazione postsinaptica con 200 μL di SDS al 5%. Sonicare il pellet, iF richiesto. </li></ol> 3. Analizzare i campioni mediante immunoblot per convalidare la frazionamento della membrana <ol><li> Determinare la quantità di proteine ​​in ciascuna frazione ( cioè le frazioni totali, extra-, pre- e post-sinaptiche) utilizzando il saggio della proteina dell&#39;acido bicinconico. </li><li> Prendere 20 μg di proteine ​​da ciascuna frazione e diluire fino ad un volume finale di 50 μl di SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Bollite per 5 min a 100 ºC. </li><li> Separare le proteine ​​mediante elettroforesi SDS-PAGE 10%, con un gel di concentrazione del 4% in condizioni riducenti. Elettroforesi a tensione costante di 80 V fino a quando il colorante entra nel gel inferiore e quindi aumenta la tensione a 120 V. </li><li> Trasferire le proteine ​​in una membrana PVDF e bloccare con IB soluzione di bloccaggio per 45 minuti a temperatura ambiente a scuotimento continuo. </li><li> Incubare la membrana durante la notte a 4 ºC con l&#39;anticorpo primario indicato ( cioè anti-SNAP-25,Anti-PSD-95, anti-sinaptofisina o anti-GPR37) diluita nella soluzione di blocco IB sotto scosse continue. </li><li> Lavare la membrana tre volte (10 min ciascuno) con la soluzione di lavaggio IB per eliminare l&#39;anticorpo primario non legato. </li><li> Incubare con l&#39;anticorpo secondario indicato per 90 min in condizioni scure a temperatura ambiente sotto scosse continue. </li><li> Lavare la membrana tre volte (10 min ciascuna) con la soluzione di lavaggio IB per eliminare l&#39;anticorpo secondario non legato. </li><li> Incubare la membrana con substrato chemiluminescente (preparare il miscuglio in condizioni scure e seguire la proporzione 1: 1 della soluzione A e B fornita dal produttore). </li><li> Analizzare la membrana in un dispositivo di rilevamento chemiluminescente. </li></ol>

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The protocol presented here constitutes a powerful biochemical tool for the study of the subsynaptic distribution of specific proteins within any brain region. However, there are some drawbacks inherent to the technique that deserve to be highlighted here. For instance, one of the main limitations is the relatively large amount of tissue needed to purify a reasonable amount of protein in order to perform the immunoblot analysis of all subsynaptic fractions. This issue might be related to the fact that synapses (i.e.,...

La trasmissione sinaptica si basa sull&#39;integrità fisica della sinapsi, un concetto che era stato previsto già nel 1897 da Foster e Sherrington 1 . Così, la comprensione della distribuzione dei componenti chiave di neurotrasmissione ( es. Canali ionici, recettori, ecc. ) È essenziale per chiarire la funzione sinaptica, sia in condizioni normali che patologiche. La microscopia elettronica (EM) ha contribuito enormemente all&#39;attuale nozione ultrastrutturale delle sinapsi prototipali del sistema nervoso centrale (CNS). In tal modo, EM ha stabilito finemente le differenze tra densità pre- e postinaptica, che sono separate da una fessura di una distanza piuttosto uniforme (~ 25 nm) 2 . È interessante notare che l&#39;apparato postinaptico presenta un ispessimento relativamente continuo, elettrone-denso, sotto la sua membrana plasmatica, la cosiddetta densità postinaptica o PSD 2 . Al contrario, all&#39;apparato presinaptico, un avvisoLa rete cytomatica discontinua è disposta appena sotto la membrana plasmatica, che è essenziale per l&#39;allineamento e l&#39;ancoraggio delle vescicole sinaptiche alla zona attiva della membrana plasmatica 3 . Quindi, EM costituisce l&#39;approccio sperimentale d&#39;oro per studiare la distribuzione delle proteine ​​all&#39;interno di sinapsi CNS strutturalmente conservati. Tuttavia, le informazioni fornite da microscopi elettronici sono statici. Infatti, le evidenze accumulate mostrano che le sinapsi in vivo sono estremamente dinamiche, provando quindi drammatiche modifiche strutturali alla trasmissione sinaptica sostenuta. Inoltre, la morfologia e la composizione delle sinapsi possono cambiare in diverse regioni del CNS e su sviluppo, maturazione, invecchiamento e sviluppo di condizioni neuropatologiche. Nel complesso, un protocollo focalizzato sull&#39;isolamento di proteine ​​appartenenti a diversi compartimenti sinaptici in condizioni fisiologiche rappresenta uno strumento prezioso per uno studio più completo del funzionamento sinaptico. <p cQui descriviamo questo tipo di approccio sperimentale complementare, che consente l&#39;arricchimento biochimico preparatorio dei diversi compartimenti sinaptici della membrana, vale a dire domini a membrana extra-, pre- e postsynaptici. Questo metodo di frazionamento della membrana, descritto per la prima volta da Philips et al . (2001) 4 , si basa su un cambiamento di pH che indebolisce le interazioni adesive che si verificano all&#39;interno dell&#39;apparato pre- e postinaptico. In primo luogo, utilizzando detergenti delicati a pH 6,0, è possibile distinguere la giunzione aderente che contiene l&#39;apparato pre- e postsinaptico e che viene mantenuto dal dominio della membrana extrasynaptica, che è solubilizzato e quindi può essere estratto dai contatti sinaptici. Successivamente, sollevando il pH da 6,0 a 8,0 in presenza di detergenti delicati, indebolisce la forza della giunzione aderente che mantiene la zona attiva presinaptica strettamente legata alla densità postinaptica. Quindi, lo scompartimento presinaptico è cosìLubrificata e può essere separata dalla densità postsinaptica, che è per lo più conservata perché la concentrazione del detergente utilizzato non promuove la sua solubilizzazione 4 . È interessante notare che l&#39;efficienza di frazionamento, eventualmente superiore al 90%, può essere confermata da diversi marcatori sottosintesi: i ) la proteina associata alla sinaptosoma 25 (SNAP-25), dalla zona attiva presinaptica; Ii ) sinaptofisina, dalla frazione extrasinaptica ( cioè, al di fuori della zona attiva e microsomi inclusi); E iii ) proteina densità postinaptica 95 (PSD-95), dalla densità postinaptica. In particolare, questo metodo di frazionamento della membrana cerebrale è stato utilizzato con successo. Di conseguenza, è stato possibile determinare con precisione la localizzazione subsynaptica di diversi recettori, quali i recettori 5 dell&#39;acido alfa-ammino-3-idrossi-5-metil-4-isossazolepropionico (AMPA), il recettore dell&#39;adenosina A1 (A 1 R) 6 ,Adenosina A 2A (A 2A R) 7 , adenosine trifosfato (ATP) P2, 8 recettori recettori del acetilcolino nicotinico 9 e recettore GPR3710 associato alla malattia di Parkinson. Tuttavia, una serie di limitazioni può ostacolare la corretta valutazione della distribuzione sinaptica di una particolare proteina neuronale. Quindi, in questa procedura, non solo descriviamo completamente l&#39;intero protocollo, ma evidenziamo anche alcuni punti critici da prendere in considerazione, come la quantità piuttosto elevata di tessuti necessari, la bassa resa proteica e il requisito obbligatorio per convalidare l&#39;efficienza Ogni separazione prima di eseguire l&#39;esperimento definito.

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	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Sucrose</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,316,211,211</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">CaCl2</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">2,112,211,210</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">MgCl2&#183;6H2O</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,313,961,210</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">Protease inhibitor cocktail Set III</td>
			<td style="width:249px;">Millipore, Darmstadt, Germany</td>
			<td style="width:160px;">535140</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Trizma Base</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">T1503</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Tris-HCl</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,236,541,209</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Triton X-100</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">X100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">SDS</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">L3771</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Glycerol</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">G5516</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Bromophenol Blue</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,311,651,604</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Dithiothreitol</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">D0632</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Tween 20</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">P2287</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Non fat dry milk</td>
			<td style="width:249px;"></td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">NaCl</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,216,591,211</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">KCl</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,314,941,210</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">KH2PO4</td>
			<td style="width:249px;">Merck</td>
			<td style="width:160px;">4873</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Na2HPO4</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,316,781,211</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Basic 20 pH</td>
			<td style="width:249px;">Crison, Alella, Spain</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">Polytron VDI 12 Adaptable Homogenizer</td>
			<td style="width:249px;">VWR, Radnor, PA, USA.</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:128px;">Ultra-Clear Tubes (14x89mm)</td>
			<td style="width:249px;">Beckman Coulter, Hospitalet de Llobregat, Barcelona</td>
			<td style="width:160px;">344059</td>
			<td style="width:167px;">Tubes should be filled almost completely when used to prevent collapsing due to ultracentrifugation.</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:128px;">Amicon Ultra-15 Centrifugal filters Ultracel -10K</td>
			<td style="width:249px;">Merck Millipore, Darmstadt, Germany</td>
			<td style="width:160px;">UFC901008</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Centrifuge 5430R</td>
			<td style="width:249px;">Eppendorf, Hamburgo, Germany</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">Optima L-90K Ultracentrifuge</td>
			<td style="width:249px;">Beckman Coulter, Hospitalet de Llobregat, Barcelona</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Sonifier 250</td>
			<td style="width:249px;">Branson, Danbury, Connecticut</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Amersham Imager 600</td>
			<td style="width:249px;">GE Healthcare Europe GmbH, Barcelona, Spain</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:128px;">Disposable Glass Pasteur Pippetes 230 mm</td>
			<td style="width:249px;">VWR, Radnor, PA, USA</td>
			<td style="width:160px;">612-1702</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">Compact Balance EK-610</td>
			<td style="width:249px;">A&#38;D, Tokyo, Japan</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">Pierce&#8482; BCA Protein Assay Kit</td>
			<td style="width:249px;">Pierce Biotechnology, Rockford, IL, USA</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:128px;">SuperSignal west pico chemiluminescent substrate</td>
			<td style="width:249px;">Thermo Fisher Scientific Inc., Waltham, MA, USA</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">GR 200 Precision Balance</td>
			<td style="width:249px;">A&#38;D, Tokyo, Japan</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:128px;">Anti-GPR37</td>
			<td style="width:249px;">Homemade antibody anti-GPR37 produced and validated in Francisco Ciruela Laboratory.</td>
			<td style="width:160px;"></td>
			<td style="width:167px;">Primary antibodies used at a final concentration of 0.250ug/ml</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:128px;">Anti-SNAP-25, anti-PSD-95, anti-synaptophysin</td>
			<td style="width:249px;">Abcam, Cambridge, United Kingdom</td>
			<td style="width:160px;"></td>
			<td style="width:167px;">Primary antibodies diluted 1:10000</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">HRP-conjugated goat anti-mouse IgG</td>
			<td style="width:249px;">Thermo Fisher Scientific, Waltham, MA, USA.</td>
			<td style="width:160px;"></td>
			<td style="width:167px;">Secondary antibody diluted 1:10000</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">HRP-conjugated goat anti-rabbit IgG</td>
			<td style="width:249px;">Thermo Fisher Scientific, Waltham, MA, USA.</td>
			<td style="width:160px;"></td>
			<td style="width:167px;">Secondary antibody diluted 1:30000</td>
		</tr>
	</tbody>
</table>

brain membrane fractionation: an ex vivo approach to assess subsynaptic protein localization

La valutazione della composizione e della funzione sinaptica della proteina costituisce una sfida importante nella neuroscienza. Tuttavia, non è facile valutare la neurotrasmissione che si verifica all&#39;interno delle sinapsi perché è altamente regolata da interazioni proteiche-proteiche dinamiche e eventi di fosforilazione. Di conseguenza, quando uno qualsiasi metodo viene utilizzato per studiare la trasmissione sinaptica, un obiettivo principale è quello di preservare queste modifiche fisiologiche transitorie. Qui presentiamo un protocollo di frazionamento della membrana cerebrale che rappresenta una procedura robusta per isolare le proteine ​​appartenenti a diversi compartimenti sinaptici. In altre parole, il protocollo descrive una metodologia biochimica per realizzare arricchimento proteico dai compartimenti presinaptici, postinaptici e extrasinaptici. In primo luogo, sinaptosomi o terminali sinaptici vengono ottenuti da neuroni che contengono tutti i compartimenti sinaptici mediante un gradiente discontinuo di saccarosio. Da notare, la qualità di questa preparazione iniziale della membrana sinaptica è critical. Successivamente, l&#39;isolamento dei diversi compartimenti sottinaptici viene raggiunto con la solubilizzazione leggera usando detergenti delicati a condizioni di pH differenziali. Ciò consente la separazione mediante gradiente e centrifughe isopiche. Infine, l&#39;arricchimento delle proteine ​​nei differenti compartimenti subsintaptici ( cioè frazioni di membrana pre-, post- ed extrasinaptiche) viene convalidato mediante analisi di immunoblot mediante marcatori proteici sinaptici ben caratterizzati ( cioè SNAP-25, PSD-95 e sinaptofisina, Rispettivamente), consentendo così una valutazione diretta della distribuzione sinaptica di qualsiasi particolare proteina neuronale.

La metodologia descritta è stata ampiamente utilizzata per l&#39;analisi subsynaptica delle proteine ​​neuronali in generale e per l&#39;isolamento e la caratterizzazione biochimica dei ricercatori sinaptici 5 , 6 , 7 , 8 , 9 in particolare. È interessante notare che il risultato rappresentativo mostrato qui mostra l&#39...

Watch this Scientific Journal Video about Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization at JoVE.com

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Qui presentiamo un protocollo di frazionamento della membrana cerebrale che rappresenta una procedura robusta per isolare le proteine ​​appartenenti a diversi compartimenti sinaptici.

Frazione di membrana del cervello: An<em&gt; Ex Vivo</em&gt; Approccio per valutare la localizzazione della proteina subsynaptica

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

brain-membrane-fractionation-an-ex-vivo-approach-to-assess

Research

JoVE Journal

Biochemistry

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

10.3K Views.  Universitat de Barcelona, L'Hospitalet de Llobregat.  Qui presentiamo un protocollo di frazionamento della membrana cerebrale che rappresenta una procedura robusta per isolare le proteine ​​appartenenti a diversi compartimenti sinaptici. 

Video: Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Utilizzando impalcatura liposomi per ricostituire interazioni proteina-proteina lipidi-prossimale<em&gt; In Vitro</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Ricerca

Biochimica

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

Rilevazione di piccolo GTPase prenilazione e GTP Binding mediante frazionamento di membrana e GTPase-collegata dell'immunosorbente

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation 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 ...

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as well as synthesis of essential metabolites. These organelles consist of the following three key sub-compartments. The envelope, characterized by two membranes, surrounds the organelle and controls the communication of the plastid with other cell compartments. The stroma is the soluble phase of the chloroplast and the main site where carbon dioxide is converted into carbohydrates. The thylakoid membrane is the internal membrane network consisting of grana (flat compressed sacs) and lamellae (less dense structures), where oxygenic photosynthesis takes place. The present protocol describes step by step procedures required for the purification, using differential centrifugations and Percoll gradients, of intact chloroplasts from Arabidopsis, and their fractionation, using sucrose gradients, in three sub-compartments (i.e., envelope, stroma, and thylakoids). This protocol also provides instructions on how to assess the purity of these fractions using markers associated to the various chloroplast sub-compartments. The method described here is valuable for subplastidial localization of proteins using immunoblotting, but also for subcellular and subplastidial proteomics and other studies.

Here, we describe a method to purify intact chloroplasts from Arabidopsis leaves and their three main sub-compartments (envelope, stroma, and thylakoids), using a combination of differential centrifugations, continuous Percoll gradients, and discontinuous sucrose gradients. The method is valuable for subplastidial and subcellular localization of proteins by immunoblotting and proteomics.

Preparazione del cloroplasto Sub-compartimenti da Arabidopsis per l'analisi di localizzazione della proteina di Immunoblotting o proteomica

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

Un'analisi di spettroscopia di fluorescenza fluttuazione delle interazioni proteina-proteina a contatti cellula-cellula

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

Valutazione delle interazioni tra proteine e proteine utilizzando una tecnica di digestione On-Membrane

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by F&#246;rster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.

This protocol describes an original setup combining spectral and fluorescence lifetime measurements to evaluate F&#246;rster resonance energy transfer (FRET) between rhodamine-based fluorescent probes and lignin polymer directly in thick plant sections.

Misurazione delle interazioni tra sonde fluorescenti e lignina nelle sezioni delle piante mediante sFLIM in base all'autofluorescenza nativa

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal transduction; from sensing environmental signals to biological response; from metabolic regulation to developmental control. Accurate structural information of protein-protein interactions is crucial for understanding the molecular mechanisms of membrane protein complexes and for the design of highly specific molecules to modulate these proteins. Many in vivo and in vitro approaches have been developed for the detection and analysis of protein-protein interactions. Among them the structural biology approach is unique in that it can provide direct structural information of protein-protein interactions at the atomic level. However, current membrane protein structural biology is still largely limited to detergent-based methods. The major drawback of detergent-based methods is that they often dissociate or denature membrane protein complexes once their native lipid bilayer environment is removed by detergent molecules. We have been developing a&#160;native cell membrane nanoparticle system for membrane protein structural biology. Here, we demonstrate the use of this system in the analysis of protein-protein interactions on the cell membrane with a case study of the oligomeric state of AcrB.

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

Sistema di nanoparticelle a membrana cellulare nativa per l'analisi dell'interazione proteina-proteina di membrana

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Polysome Profiling without Gradient Makers or Fractionation Systems

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs being translated by ribosomes, and analyze polysome associated factors. While automated gradient makers and gradient fractionation systems are commonly used with this technique, these systems are generally expensive and can be cost-prohibitive for laboratories that have limited resources or cannot justify the expense due to their infrequent or occasional need to perform this method for their research. Here, a protocol is presented to reproducibly generate polysome profiles using standard equipment available in most molecular biology laboratories without specialized fractionation instruments. Moreover, a comparison of polysome profiles generated with and without a gradient fractionation system is provided. Strategies to optimize and produce reproducible polysome profiles are discussed. Saccharomyces cerevisiae is utilized as a model organism in this protocol. However, this protocol can be easily modified and adapted to generate ribosome profiles for many different organisms and cell types.

This protocol describes how to generate a polysome profile without using automated gradient makers or gradient fractionation systems.

Profilazione dei polisomi senza generatori di gradienti o sistemi di frazionamento

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs ...

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards.

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances, many yeast mitochondrial proteins still remain with their functions completely unknown. This protocol provides a simple and reliable method to determine the submitochondrial localization of proteins, which has been fundamental for the elucidation of their molecular functions.

Valutazione della localizzazione della proteina submitocondriale nel lievito in erba Saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...