This work was supported by NIH grant GM085232 and an Irma T. Hirschl/ Monique Weill-Caulier Scholar Award (to A.A.).

1. Lipid Preparazione <ol><li> Aliquota lipidi quantità desiderata in un tubo di vetro trasparente. Vedi. coli estratto lipidico polare, ma la maggior parte delle composizioni lipidiche possono essere usati. Se i lipidi sono in forma di polvere, risospendere in cloroformio ad una concentrazione di 20 mg / ml. Asciugare i lipidi a RT in N 2 gas fino a quando tutto il solvente è evaporato. </li><li> Risospendere i lipidi in pentano e asciugare nuovamente un flusso costante di gas N 2 per eliminare tracce residue di cloroformio. Dry fino a che tutto il solvente è evaporato. Mentre l&#39;essiccazione, ruotare delicatamente il tubo in modo che il lipide è distribuito in una pellicola uniforme che copre la parte inferiore del tubo piuttosto che formare una densa massa sul fondo. </li><li> Aggiungere il tampone di ricostituzione contenente detersivo (KCl 300 mm, 50 acido citrico mM, 25 mm K 2 HPO 4, pH 4.5, 35 CHAPS mm) per sciogliere i lipidi. Utilizzare altri detergenti delicati per questo passo se la proteina è scarsamente tollerante CHAPS. </li><li> Resuspend i lipidi di chiarezza con un sonicatore bagnomaria. Immergere il tubo nel centro del bagno sonicatore acqua alla potenza massima breve (30 sec-1 min) con impulsi brevi (30 sec-1 min) interrompe. La soluzione dovrebbe diventare chiaro. NOTA: Il grado finale di chiarezza raggiunto dipende dalla composizione lipidica specifico utilizzato. Alcune variazioni da lotto a lotto in questa fase, anche tra lipidi nominalmente identici, è anche possibile. Haze residuo è dovuta alla dispersione della luce da grandi particelle in sospensione, che può essere rimosso mediante centrifugazione. </li><li> Incubare i lipidi risospeso a RT per 20-60 min. </li></ol> 2. Proteoliposome Formazione NOTA: Diverse strategie può essere impiegato per inserire la proteina detergente solubilizzato in liposomi. Per CLC-EC1 dialisi funziona bene ed è quindi il metodo di scelta 6,9,10. <ol><li> Aggiungere la proteina purificata alla desiderata proteina-to-lipidi (P / L) Rapporto (espressa in mg o<html> f Proteine ​​/ mg lipidi). La scelta del rapporto P / L dipende dallo scopo dell&#39;esperimento. <ol><li> Se l&#39;obiettivo è quello di valutare l&#39;attività della proteina di interesse, ricostituire ad alta P / L&#39;s per massimizzare il segnale, in quanto ogni liposoma contiene più copie della proteina. Per quantificare l&#39;attività e / o velocità di trasporto unitario della proteina, ricostituire in un regime di diluizione Poisson a bassa P / L&#39;s, in modo che ogni vescicola contiene in media 1 proteine. Vedere il punto 7 e discussione per una più approfondita analisi di quando usare ogni regime. </li></ol></li><li> Per determinare i valori ottimali P / L per i vari regimi, effettuare una titolazione completa dell&#39;attività in funzione della concentrazione proteica 8,11-13. Tuttavia, per ogni proteina per la quale il peso molecolare (MW, espresso in kDa) dell&#39;unità attiva è noto i seguenti valori P / L possono essere utilizzati come guesstimates iniziali: pg &quot;/&gt; <img alt="Equazione 2" fo:content-width="1.5in" src="/files/ftp_upload/52369/52369eq2.jpg" /></li><li> Caricare la miscela di proteine ​​e lipidi in un tubo di dialisi pre-bagnata o una cassetta. Posizionare il dispositivo di dialisi in un becher contenente 500-1,000x il volume del buffer lipidi ricostituzione (cioè, 1 tampone L per 1 ml di lipidi risospensione) sotto leggera agitazione. <ol><li> Change tampone 3-4 volte a intervalli&gt; 3 ore. Ad ogni cambio soluzione, lavare la cassetta / tubo e il bicchiere con acqua distillata e riempire il bicchiere con tampone di ricostituzione fresca. Includere 1-2 O / N intervalli per il cambio soluzione. Il numero esatto e durata delle modifiche soluzione dipende lipide esatta e detergenti utilizzati. </li></ol></li><li> Dopo l&#39;ultimo passo, rimuovere i liposomi dalla nave di dialisi, le aliquote in tubi e flash li congelare in N liquido 2 o congelamento a -80 ° C. </li></ol> 3. Registrazione Set-up &lt;p class = &quot;jove_content&quot;&gt; NOTA: La registrazione set-up (Figura 2A) si compone di due camere (cilindri a fondo piatto, ~ 3-4 volumi ml), un Cl - (vedi sotto) elettrodo, un pH-metro con un analogo o uscita digitale elettrica, un digitalizzatore, e un computer con un appropriato software di acquisizione. <ol><li> Collegare il Cl - elettrodo al pH-metro, l&#39;uscita del misuratore di pH al digitalizzatore, che è collegata al computer. Inserire l&#39;elettrodo di riferimento in una camera e il filo ricodifica nell&#39;altro (Figura 2B). NOTA: La polarità dei fili è corretto se ΔV Cal (vedi punto 6.4 e 7.2) è positivo. </li><li> Mettere le camere su un piatto mescolare con un ancoretta in camera di registrazione (Figura 1B). Assicurarsi che la ancoretta non tocchi l&#39;elettrodo. </li><li> Collegare camere utilizzando un ponte di agar (Figura 2B) (KCl 100 mM e 2% agarosio). Conservare i ponti Agar in KCl 100 mm. </li&gt; </ol> 4. Preparazione del Cl - Elettrodo <ol><li> Prendete un vecchio e -possibly- elettrodo non funzionante pH. Rompere il rivestimento di vetro per rivelare completamente i fili d&#39;argento. </li><li> Rimuovere delicatamente il rivestimento dai fili d&#39;argento con una lama di bisturi. Pulire i fili con abbondanti quantità di H 2 O e EtOH. </li><li> Mettere in una soluzione satura di FeCl 3 (o candeggina) fino fili sono coperti con un mantello scuro uniforme di AgCl. </li></ol> 5. Preparazione di unilamellari vescicole <ol><li> La notte prima degli esperimenti, si gonfiano 1 g di Sephadex G-50 perle in 15 ml di tampone esterna (1 mM KCl, 150 mm K 2 SO 4, acido citrico 25 mM, pH 4.5) con agitando delicatamente (non mescolare) per almeno 3 ore a temperatura ambiente prima dell&#39;uso. Ogni esperimento richiede ~ 3 ml di perle gonfie. Mantenere le perle gonfie a 4 ° C per pochi giorni al massimo. </li><li> Mentre i liposomi sono scongelamento a RT, versare in ogni colonna ~ 3 ml di gonfiore G-50 perline. Lasciate asciugare per flusso di gravità a RT; questo richiede di solito 1-2 min. </li><li> Preparare vescicole unilamellari estrudendo i proteoliposomi 11 volte attraverso un mini-estrusore utilizzando un taglio 0,4 m Teflon. </li><li> Porre la colonna in un tubo a fondo tondo di plastica. Gira le colonne per rimuovere la soluzione in eccesso. Centrifugare per 20-30 secondi a 1.400 xg in una centrifuga clinica. </li><li> Scartare flow-through, colonna posto in un tubo di vetro 13 x 100 mm e aggiungere 100 ml di vescicole estrusi alla colonna. Spin colonna per 1 min a 500 xg in una centrifuga clinica. </li><li> Raccogliere ~ 200 ml di flusso continuo che verranno aggiunti alla camera di registrazione al punto 6.5. </li></ol> 6. Efflux misura <ol><li> 2 ml di KCl 100 mm di riferimento (terra) da camera e 1,8 ml del tampone esterna (1 mM KCl, 150 mm K 2 SO 4, 25 acido citrico mM, pH 4,5) per la camera di registrazione. Il leggero squilibrio osmotico tra l&#39;internoe soluzioni esterne non influisce </li><li> Avviare il programma di acquisizione. Lasciate che la linea di base equilibrare, l&#39;operazione potrebbe richiedere alcuni minuti. </li><li> Una volta che il segnale raggiunge una linea di base stabile (liposomi sono pronti e le colonne asciugare), avviare la registrazione (Figura 3A). </li><li> Aggiungere 15 ml di una soluzione 10 mM KCl per calibrare il sistema (Figura 2A). </li><li> Aggiungi liposomi. Un piccolo salto potrebbe essere visibile a causa della rimozione incompleta di Cl esterni - dalle proteoliposomi (Figura 3A). Aspetta la linea di base si stabilizza. </li><li> Aggiungere Valinomicina (1 ml a 1 mg / ml in EtOH) per avviare efflusso (Figura 3A). </li><li> Lasciate che l&#39;efflusso suo corso tempo fino a che altipiani (Figura 3A). </li><li> Aggiungere 40 ml di 1,5 M β-octylglucoside (β-OG) preparato nel buffer esterna per sciogliere tutti liposomi (Figura 3A). Terminare la registrazione. </li></ol> 7. Dati AANALISI <ol><li> Esportare il file di traccia da in formato ASCII o testo e importarlo nel programma di analisi di scelta. </li><li> Misurare la tensione nei seguenti punti (Figura 3A): All&#39;inizio della registrazione (V 0); Dopo aggiunta dell&#39;impulso di taratura (V cal); Dopo l&#39;aggiunta dei liposomi (V lipo); Alla fine del efflusso (V fin); Dopo l&#39;aggiunta di detergente (V tot); Baseline le tensioni in modo che V 0 = 0. </li><li> Utilizzare l&#39;equazione di Nernst-Plank per determinare il valore sperimentale <img alt="Equazione 3" fo:content-width=".3in" src="/files/ftp_upload/52369/52369eq3.jpg" /> misurando <img alt="Equazione 4" fo:content-width="3in" src="/files/ftp_upload/52369/52369eq4.jpg" /> Dove Vol Cal è il volume dell&#39;impulso di taratura in ml, 1.800 è il volume della camera all&#39;inizio dell&#39;esperimento espressa1; l, [Cl] cal / a sono il Cl - concentrazioni di impulso di calibrazione e del cuscinetto esterno in mM e ΔV cal è il salto in mV misurata dopo l&#39;impulso di taratura. NOTA: Questa determinazione empirica di α tre scopi: garantisce che il sistema risponde correttamente, offre una misura della coerenza della risposta dello strumento tra esperimenti e controllare che errori sono stati fatti nella fabbricazione soluzione. </li><li> Calcolare le concentrazioni Cl dopo ogni passaggio come segue: <img alt="Equazione 5" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq5.jpg" /> <img alt="Equazione 6" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq6.jpg" /> <img alt="Equazione 7" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq7.jpg" /> Il fattore 3/400 proviene dalla diluizione dell&#39;impulso di taratura 15 microlitri nel volume finale di 2000 ml. </li><li> Calculmangiato i cambiamenti [Cl] ad ogni passo, ΔCl lipo / def / tot. </li><li> Convertire V (t) in Cl (t) con <img alt="Equazione 8" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq8.jpg" /> Direttamente determinare i valori [Cl] nei punti critici e confrontarle con i valori calcolati come controllo interno. </li><li> Normalizzare la traccia in modo che la lipo Cl rel = 0 e Cl rel tot = 1 <img alt="Equazione 9" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq9.jpg" /></li><li> Montare corso tempo di efflusso la seguente equazione: <img alt="Equazione 10" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq10.jpg" /> Quando L è il tasso di Cl - perdita che è sperimentalmente determinata misurando Cl - efflusso da liposomi privi di proteine ​​preparati nelle stesse condizioni e τ è la costante di tempo del processo. </li><li> Calcolare v, la velocità iniziale: Dove ΔCl pinna è espresso in millimolare e V è il volume della camera in microlitri. </li><li> Calcolare il tasso di trasporto / conduttanza <img alt="Equazione 12" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq12.jpg" /> Dove P è il P / L, MW è il peso molecolare del complesso attivo espresso in kDa </li></ol>

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The authors declare no competing financial interests.

Abbiamo descritto un protocollo dettagliato per misurare Cl - il trasporto mediato da canali anione selettivo purificati o trasportatori ricostituiti in liposomi. L&#39;esempio è stato utilizzato il procariote H + / Cl - Scambiatore CLC-EC1. Tuttavia, il metodo può essere facilmente adattato per studiare canali gated da ligandi 12,13,15, 11,12 tensione, o sportive diversa selettività anionico 15,16 sostituendo l&#39;Ag: Elettrodo AgCl con uno adatto ione in esame...

Negli ultimi due decenni la capacità di iperespressione e purificare proteine ​​di trasporto di membrana è drammaticamente aumentato: i canali ionici, trasportatori primari e secondari sono ormai di routine purificati da sistemi di espressione eterologhi e fonti naturali. Nuovi approcci per monitorare l&#39;espressione, di migliorare e facilitare l&#39;estrazione e migliorare la stabilità di queste proteine ​​sono in continua evoluzione 1-5. Queste innovazioni tecnologiche sono state fondamentali per innescare l&#39;esplosione di livello atomico informazioni sulla struttura delle proteine ​​di membrana che, a sua volta, migliorato la nostra comprensione delle basi strutturali della loro funzione. Al contrario, la nostra capacità di sondare le proprietà funzionali delle proteine ​​purificate non aumenta alla stessa velocità, in modo che in alcuni casi alta risoluzione informazione strutturale è accompagnato da dati qualitativi funzionali, limitando così la possibilità di verificare quantitativamente predizioni basati sulla struttura. Quindi, la SVILUPPOt di saggi funzionali quantitativi e generalizzabili è un passo fondamentale verso la delucidazione delle basi meccanicistiche della funzione delle proteine ​​di membrana. Qui si descrive un saggio di efflusso che può essere usato per determinare quantitativamente importanti proprietà funzionali purificati e ricostituiti Cl - canali e trasportatori. I principi alla base del dosaggio può essere generalizzati ad una varietà di sistemi di trasporto, nonché proteine ​​non-ionici trasporto. I liposomi vengono ricostituiti con purificati Cl - canale / trasportatori in presenza di un grande Cl - gradiente (Figura 1A, B). Cl - efflusso viene avviata mediante l&#39;aggiunta di uno ionoforo per consentire flusso contro-ione, nel nostro caso il K + ionoforo valinomicina, che shunt la tensione stabilita dal Cl - gradiente e impostare il potenziale di membrana iniziale al potenziale di equilibrio di K + 6,7. Senza tegli ionoforo significative netto Cl - verifica efflusso, in quanto è impedita dalla generazione di un potenziale transmembrana. I dati sono quantitativamente descritto da due parametri misurabili (Figura 1C): τ la costante di tempo di Cl - efflusso, e f 0, la frazione di liposomi contenenti una proteina attiva. Da τ e f 0 unitario Cl - velocità di trasporto, la frazione di proteine ​​attive e la massa molecolare del complesso attivo possono essere derivate 8. La tecnica è illustrata qui utilizzando proteoliposomi ricostituiti con CLC-EC1, un ben caratterizzato CLC-type H + / Cl - scambiatore di struttura e funzione nota. Questo saggio è facilmente generalizzabile a canali o trasportatori con selettività ionica differente o la cui attività dipende dalla presenza di tensione e / o leganti. Inoltre, questo test può essere utilizzato per determinare se piccole molecole influenzano direttamente la proteina ricostituito,per quantificare gli effetti di questi composti e come composizione membrana o reagenti ipolipemizzanti influiscono sulla funzione dei canali ricostituiti e trasportatori.

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			<td height="21" style="height:21px;width:372px;">Name of Reagent/ Equipment</td>
			<td style="width:337px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liposomicator, Avanti Polar Lipids Inc.</td>
			<td style="width:337px;">&#160;Avanti Polar Lipids Inc.</td>
			<td>610200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IEC Centra CL2 Benchtop</td>
			<td style="width:337px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Orion Research Model 701A Digital pH-mV meter</td>
			<td style="width:337px;"></td>
			<td style="width:119px;"></td>
			<td>These can be found on Ebay.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Non-functional pH probe</td>
			<td style="width:337px;"></td>
			<td style="width:119px;"></td>
			<td>Any pH meter probe with silver wires will work. The glass/plastic coating needs to be removed and the wires cleaned.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160; DI-710 Data Logger</td>
			<td style="width:337px;">DATAQ instruments</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">WinDAQ acquisition software</td>
			<td style="width:337px;">DATAQ instruments</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pierce Disposable Plastic Columns, Gravity-flow, 2ml</td>
			<td style="width:337px;">Pierce (Thermo Scientific)</td>
			<td>29922</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KIMAX Culture Tubes, Disposable, Borosilicate Glass</td>
			<td style="width:337px;">Kimble Chase</td>
			<td>73500-13100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Extruder Set With Holder/Heating Block</td>
			<td style="width:337px;">Avanti Polar Lipids Inc.</td>
			<td align="right" style="width:119px;">610000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Computer</td>
			<td style="width:337px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
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</table>

a proteoliposome-based efflux assay to determine single-molecule properties of cl- channels and transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl- from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl- exchanger, as a model system. The efflux assay can be utilized to study any Cl- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

Descriviamo un protocollo dettagliato e robusto per misurare Cl - il trasporto mediato da purificato CLC-EC1, un procariotico CLC-tipo H + / Cl - scambiatore, ricostituito in liposomi. Una rappresentazione schematica dell&#39;esperimento è mostrato in Figura 3 proteoliposomi ricostituiti con purificato CLC-CE1 e con alta Cl interna -. Sono immersi in una soluzione bagno contenente bassa Cl -. In queste condizioni net Cl - efflusso è imp...

Watch this Scientific Journal Video about A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters at JoVE.com

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

A Proteoliposome-Based Efflux Assay per determinare le proprietà singola molecola di Cl<sup&gt; -</sup&gt; Canali e trasportatori

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...

a-proteoliposome-based-efflux-assay-to-determine-single-molecule

Research

JoVE Journal

Biology

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

9.7K Views.  Weill Cornell Medical College. Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

Video: A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

Metodi di singola molecola per monitorare l&#39;evoluzione a doppio strato: Proprietà meccaniche

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

Ricerca

Biologia

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

Visualizzazione singola molecola di DNA replica con microscopia a fluorescenza

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

Osservazione diretta del DNA Enzimi Replica utilizzando un singolo-molecola di DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

Singola molecola Imaging dei trasporti nucleari

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

Un test di screening a fluorescenza per l&#39;identificazione di modulatori Canali GIRK

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

Combinando singola molecola di manipolazione e di imaging per lo studio delle interazioni proteina-DNA

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

Studiare Looping DNA a singola molecola FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Misura e Analisi di extracellulare Acido prodotte per determinare glicolitica Tasso

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

The Lactate Dehydrogenase Sequestration Assay &amp;#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

Il dosaggio di sequestro di lattato deidrogenasi — Un metodo semplice e affidabile per determinare l'attività autofagica sequestro Bulk in cellule di mammifero

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

Analisi cinetica ad alta velocità ad alto rendimento parallela per cleavage del DNA specifico del sito

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...