Il lavoro in questo manoscritto è stato sostenuto da fondi del Dipartimento di Radioterapia Oncologica e Huntsman Cancer Institute e il National Institute of Health sovvenzione (R01-CA188520) di SB. Ringrazio Danielle Johnson e Steven Bennett nel mio laboratorio per dimostrare il protocollo e spiegando i suoi benefici. Sono grato al Dr. Mahesh Chandrasekharan (Huntsman Cancer Institute) per i commenti critici sul manoscritto.

1. immunoprecipitazione della cromatina (ChIP) in seguito BrdU etichettatura <ol><li> Culture cellule NIH3T3 in presenza sia di DMSO o inibitori selettivi HDAC1,2 come descritto in precedenza 6. </li><li> Dopo il trattamento con inibitori selettivi DMSO o HDAC1,2, aggiungere BrdU alle celle in una cappa coltura tissutale sterile. Incubare 2 x 10 6 cellule NIH3T3 placcati in 10 ml mezzi NIH3T3 con una concentrazione finale di 20 mM di bromodeossiuridina (BrdU) per 60 min in una sterile 37 ° C coltura tissutale incubatore. </li><li> Crosslink proteine ​​intracellulari DNA aggiungendo formaldeide direttamente al mezzo di coltura ad una concentrazione finale di 1%. Incubare 2 x 10 6 cellule NIH3T3 con formaldeide a temperatura ambiente per 10 min su un agitatore. </li><li> Quench la reticolazione mediante l&#39;aggiunta di glicina ad una concentrazione finale di 125 mM. Incubare a temperatura ambiente per 5 minuti su un agitatore. </li><li> Togliere la carta e raccogliere le cellule in PBS in una provetta da centrifuga da 15 ml. Spin cells a 956 xg per 5 minuti a 4 ° C. Lavare il pellet di cellule due volte con PBS freddo. Rimuovere PBS dal tubo. Conservare il pellet reticolato cellulare senza PBS (surnatante) a -80 ° C fino al momento dell&#39;uso. </li><li> Preparare il lisato dal pellet cellulare reticolato aggiungendo tampone FA140 500 microlitri (50 mM di HEPES KOH pH 7.5, 140 mM di NaCl, 1 mM di EDTA (pH 8,0), 1% Triton-X-100 e 0,1% sodio desossicolato) integrato con inibitore della proteasi cocktail. </li><li> Taglio la cromatina dalle sonicazione dei campioni cinque volte al potere il 35% e con un impulso di 15 secondi, 0,9 secondi acceso e 0,1 secondi spento impostazione. Tenere le provette in ghiaccio dopo ogni turno di ultrasuoni per evitare il riscaldamento dei campioni. <ol><li> Controllare il taglio su un gel DNA come l&#39;efficienza di taglio varierà tra i diversi sonicatori. Per controllare sonicazione, invertire reticolazione con l&#39;aggiunta di NaCl ad una concentrazione di 0,16 M e eseguire i passaggi 1.17.1. - 1.22. Esegui eluita 10 microlitri DNA su un gel di agarosio 1,5% per verificare se il taglio ètra 300-700 bp con una intensità media a 500 bp. </li></ol></li><li> Dopo sonicazione, centrifugare i campioni per 10 min a 17.949 xga 4 ° C e trasferire il surnatante in una nuova provetta. <ol><li> Eseguire la stima delle proteine ​​utilizzando il kit di analisi delle proteine ​​commerciale secondo il protocollo del produttore. Utilizzare la stessa quantità di lisato per il chip di controllo e campioni sperimentali. In generale, utilizzare 1 mg di lisato totale per reazione chip. </li></ol></li><li> Preparare 50% sospensione di proteina A-agarosio utilizzando tampone FA140 contenente inibitori delle proteasi cocktail. Aggiungere 1/10 volume del cocktail inibitore della proteasi. Calcolare il volume di perline necessari in base al numero di campioni in totale. <ol><li> Lavare perline Proteina A-agarosio con tampone FA140 due volte per rimuovere il tampone di conservazione e aggiungere un volume uguale di tampone FA140 ai talloni per fare 50% slurry. </li><li> Per rimuovere le proteine ​​che non si legano specificamente ai talloni, aggiungere 50 ml di 50% sospensione di Proteina A-agarosio e incubate i campioni a 4 ° C con rotazione costante end-over-end in un mixer rosticceria per 30 min. </li></ol></li><li> Spin i campioni a 956 xg a 4 ° C per un minuto. Raccogliere il surnatante ed eseguire immunoprecipitazione tramite l&#39;aggiunta di 8 microlitri H4K16ac anticorpo o 5 ml (5 mg) di 1 mg / ml di anticorpo SMARCA5 il surnatante. Incubare a 4 ° CO / N con rotazione end-over-end costante in un mixer rosticceria. </li><li> Salva 1/10 ° di un estratto per il controllo &#39;input&#39; e impostarlo come parte a -20 ° C. L&#39;ingresso deve essere inverso reticolato con i campioni IP. </li><li> Utilizzare la stessa quantità di IgG di coniglio (5 ml di 1 mg / ml) come campione di controllo negativo immunoprecipitazione. </li><li> Il giorno seguente, aggiungere 50 ml di 50% proteina A-agarosio slurry all&#39;estratto e incubare per 1 ora a 4 ° C con rotazione. </li><li> Pellet agarosio per centrifugazione a 956 xg per 2 minuti a 4 ° C, rimuovere con attenzione il surnatante e wash le perline con i seguenti buffer in sequenza. Nelle preparazioni tampone, aggiungere inibitori della proteasi a destra prima dell&#39;uso. <ol><li> Lavare con sale basso Immune Complex tampone di lavaggio (FA140; concentrazione finale di 50 mM di HEPES KOH pH 7.5, 140 mM di NaCl, 1 mM di EDTA (pH 8,0), 1% Triton-X-100 e 0,1% desossicolato di sodio) per 5 minuti a 4 ° C con rotazione end-over-end. </li><li> Lavare con Salt Immune Complex tampone di lavaggio ad alta (FA500; concentrazione finale di 50 mM di HEPES KOH pH 7.5, 500 mM di NaCl, 1 mM di EDTA (pH 8,0), 1% Triton-X-100 e 0,1% desossicolato di sodio) per 5 minuti a 4 ° C con rotazione end-over-end. </li><li> Lavare con LiCl Immune Complex tampone di lavaggio (concentrazione finale di 10 mM di Tris-Cl pH 8,0, 250 mM di LiCl, 0,5% e 0,5% NP40 desossicolato di sodio) per 5 minuti a 4 ° C con rotazione end-over-end. </li></ol></li><li> Dopo lavaggi come descritto sopra, aggiungere 200 microlitri soluzione di eluizione (concentrazione finale di 1% SDS e 100 mM sodio bicarbonato) e incubate a temperatura ambiente per 15 min con rotazione end-over-end a RT. </li><li> Spin i campioni a 956 xg a RT e trasferire l&#39;eluato in una nuova provetta da 1,5 ml. Ripetere il passaggio 1.15 con l&#39;aggiunta di soluzione di eluizione 200 l. </li><li> Per 400 ml di eluato, aggiungere NaCl ad una concentrazione di 0,16 M e mescolare bene. <ol><li> Inoltre, aggiungere la soluzione di eluizione 400 microlitri di 10 microlitri di ingresso che è stato allocato (passo 1.11) e aggiungere NaCl ad una concentrazione di 0,16 M di invertire campioni di ingresso reticolazione pure. Invertire reticolare i campioni incubando a 65 ° C bagnomaria per 5 ore. </li></ol></li><li> Aggiungere 1 ml di 100% EtOH al eluato reverse-reticolato. Mescolare bene e incubare a -80 ° C per 2-3 ore oppure a -20 ° CO / N. Spin i campioni a 17.949 xg per 15 minuti e scartare l&#39;etanolo. </li><li> Aggiungere 800 ml 70% di etanolo per lavare il pellet. Spin a 17.949 xg per 15 min a 4 ° C e scartare etanolo. Spin brevemente per rimuovere qualsiasi residuo di etanolo. Asciugare i campioni a 37 ° C per 10 min per rimuovere l&#39;etanolo residuo. </li><li> Sciogliere in 90 microlitri DNA RNasi e DNasi acqua libera e aggiungere 2 microlitri 10 mg / ml RNasi a 0,2 mg / ml concentrazione finale. Incubare a 37 ° C a bagnomaria per 30 minuti. </li><li> Aggiungere 10 ml 10x Proteinase K Buffer (100 mM Tris-Cl pH 8.0, 50 mM EDTA pH 8.0, 5% SDS). Mescolare bene e aggiungere 1 ml proteinasi K. Incubare a 42 ° C per 1 ora. </li><li> Purificare ingresso e ChIP DNA usando commerciale kit di purificazione PCR secondo il protocollo del produttore ed eluire il DNA in 50 microlitri acqua. Conservare DNA a -20 ° C fino all&#39;utilizzo. </li></ol> 2. Slot blot analisi del DNA ChIP <ol><li> Misurare la resa di ingresso e di DNA ChIP eluito in 50 microlitri acqua come descritto al punto 1.22 utilizzando uno spettrofotometro a 280 nm secondo il protocollo del produttore. </li><li> Prendere volumi uguali (50 microlitri) di ingresso o di DNA immunoprecipitato che si trovano all&#39;interno della gamma lineare di rilevamento. <ol><li> Ad esempio, se l&#39;ingressoResa del DNA è di 1,5 ng / ml, poi prendere 30 ml (o 45 ng di DNA totale) e fare volume di 50 ml con acqua. Per fattore ChIP, prendere l&#39;intero 50 microlitri eluato dal punto 2.1 e passare al punto 2.3. </li></ol></li><li> Denaturare 50 microlitri di ingresso o DNA immunoprecipitato aggiungendo 2,5 volumi di 0,4 N NaOH, vortex brevemente e centrifugare per 956 xg per 10 sec, e incubare a temperatura ambiente per 30 min. </li><li> Neutralizzare il DNA denaturato con l&#39;aggiunta di 175 ml di 1 M Tris-HCl, pH 6,8, vortex, centrifugare per 956 g per 10 secondi e posizionare i campioni sul ghiaccio. </li><li> Preparare una diluizione seriale di ingresso e di DNA immunoprecipitato per la Slot Blot test come descritto di seguito. <ol><li> Seguire i passaggi 2,3-2,4 per ottenere un volume totale di 350 microlitri del campione (ad esempio, 50 ml di ingresso / DNA ChIP, 125 ml 0,4 N NaOH, 175 ml 1 M Tris-HCl, pH 6,8). Nota: Per l&#39;istanza indicato in precedenza, 45 ng di DNA ingresso finirà per essere 0,129 ng / ml. </li><li> Per il DNA di ingresso, etichettare tre tubos come 100, 50 e 25 e aggiungere 100 ml, 50 ml e 25 ml di campione di DNA denaturato e neutralizzato. Rendere il volume finale a 100 ml con acqua priva di nucleasi. Per DNA chip, label tre tubi da 200, 100 e 50 e aggiungere 200 microlitri, 100 microlitri e 50 ml di campione di DNA denaturato e neutralizzato. Fai volume finale a 200 ml con acqua priva di nucleasi. </li></ol></li><li> Tagliare la membrana alla dimensione dell&#39;apparato Slot Blot. Pre-bagnare la membrana e carta assorbente in 20x SSC prima e prima di aggiungere DNA. Assemblarle nell&#39;apparato macchia slot secondo il protocollo del produttore. </li><li> DNA pipetta dal punto 2.5.2 in apposite cave. Applicare il vuoto lentamente per tirare il DNA sulla membrana zeta-sonda. Arrestare vuoto dopo tutti i campioni sono passati attraverso l&#39;apparecchio. <ol><li> Smontare l&#39;apparato e immobilizzare immediatamente il DNA sulla membrana utilizzando un reticolante UV. Avere il lato del DNA nella reticolante UV e utilizzare il autocrosslink impostazione nel reticolante. Seguire il protocollo del produttore. </li></ol></li><li> Rileva BrdU sulla macchia slot eseguendo western blotting. <ol><li> Bloccare la membrana con 5% di latte secco non grasso in PBS contenente 0,1% Tween-20 (PBST) per 1 ora a RT per prevenire il legame non specifico degli anticorpi alla membrana. </li><li> Aggiungere anticorpo primario anti-BrdU (diluizione 1: 500) diluito nel latte secco non grasso 1% in PBST e incubare il blot con l&#39;anticorpo primario per 3 ore a temperatura ambiente su un agitatore. Lavare la membrana con PBST tre volte dopo l&#39;incubazione anticorpo primario. </li><li> Aggiungere anticorpo secondario anti-topo HRP-coniugato alla macchia (1: 5.000 diluizione) e incubare a temperatura ambiente per 1 ora. Lavare la membrana con PBST tre volte dopo l&#39;incubazione anticorpo secondario. NOTA: utilizzare un volume sufficiente di anticorpi per coprire completamente la membrana durante l&#39;incubazione anticorpi primari e secondari. </li></ol></li><li> Preparare la miscela ECL e aggiungerlo alla ac macchiacondo protocollo del produttore. Incubare la membrana con mix ECL per 5 minuti a RT. <ol><li> Posizionare la membrana in una cassetta a raggi X, esporre la membrana alla pellicola a raggi X e sviluppare il blot usando il protocollo del produttore. </li><li> Quantificare il segnale ottenuto per l&#39;ingresso e il DNA chip dopo la scansione le macchie utilizzando il software Image J secondo il protocollo del produttore. Per la quantificazione, utilizzare il segnale che è all&#39;interno del range lineare di rilevamento al fine di garantire la precisione della misurazione. </li></ol></li></ol>

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I have no competing financial interests.

Il protocollo descritto in questo manoscritto è un metodo relativamente veloce per dimostrare la presenza di proteine ​​o loro forme post-traduzionali modificati sul DNA appena replicato o nascente. Inoltre, questa tecnica permette uno per misurare la cinetica di associazione-dissociazione di una proteina o la sua forma modificata con il DNA nascente. Questa tecnica è complementare alla elegante tecnologia iPOND 13. Nella tecnologia iPOND, il DNA di nuova sintesi è etichettato con deossiuridina etile (...

Riparazione del DNA difettosa e / o replicazione del DNA sono una delle principali cause di instabilità genomica, che può innescare la morte cellulare. Un singolo non riparato doppia interruzione filo è sufficiente a causare la morte delle cellule 1. Organizzazione della cromatina è transitoriamente alterata sia in fase di replica e riparare 2,3, e l&#39;incapacità di mantenere le informazioni epigenetiche nel corso di questi processi si tradurrà in una minaccia per l&#39;integrità del genoma. Perdita di HDAC3 o della funzione HDAC1,2 impedisce progressione fase S, la replicazione e riparazione del DNA che porta a stress genotossico (danni al DNA) e la morte delle cellule 4-9. Si tratta quindi di una strategia pratica per usare inibitori HDAC selettivi di interrompere la replica, la riparazione e la cromatina in cellule tumorali e causare danni al DNA, che a sua volta può fermare la crescita e induce la morte cellulare selettivamente nelle cellule tumorali in rapida crescita. Durante la replicazione del DNA, cromatina è rapidamente smontato e poi rimontato seguendo duplicazione del DNA. Recentemente synthesized istone H4 è acetilato a K5 e residui K12 (H4K5K12ac) e segue la deposizione sulla cromatina, sono rapidamente deacetilata da istone deacetilasi (HDAC) 10,11. Fallimento di cellule per mantenere l&#39;integrità della cromatina nascente da istoni deacetilazione può portare al collasso della forcella, che a sua volta può causare danni al DNA e la morte cellulare. Abbiamo recentemente dimostrato che l&#39;inibizione selettiva di HDAC1,2 aumenta l&#39;acetilazione degli istoni (H4K5ac, H4K12ac e H4K16ac) e inibisce l&#39;attività SMARCA5 cromatina remodeler sulla cromatina nascente, che correla con una ridotta progressione forca di replicazione, aumentato crollo forchetta e aumentato la replica da stress danno al DNA 6 . Così, HDAC inibitore trattamento possono alterare la struttura della cromatina nascente per innescare danni al DNA e la morte molto rapidamente nelle cellule tumorali, in quanto questi ciclo cellule in rapida e passare più volte attraverso la fase S. E &#39;quindi importante capire come funzionano HDAC regolare acetilazione degli istoni e proteine ​​binding per mantenere la replicazione del DNA nelle cellule di mammifero. Per misurare quantitativamente la quantità di acetilazione degli istoni e SMARCA5 remodeler cromatina associata con il DNA nascente durante la replicazione del DNA, abbiamo ideato un saggio ChIP modificato chiamato la tecnica di BrdU-Chip-Slot-occidentale. Dopo immunoprecipitazione della cromatina (ChIP) di una proteina o istone modifica desiderata, la quantità di DNA nascente (all&#39;etichetta analogico timidina, BrdU) nel campione chip può essere rilevato mediante analisi Western di DNA chip BrdU marcato trasferite su una membrana utilizzando un Slot Apparecchio Blot. Utilizzando questa tecnica, abbiamo dimostrato che H4K16ac (un marchio coinvolta nella confezione cromatina) e il membro della famiglia ISWI SMARCA5 (regolatore actina-dipendente matrice associata SWI / SNF-correlata della cromatina) remodeler cromatina sono associati con il DNA nascente in cellule in fase S. 6. Abbiamo anche trovato che il marchio nascente H4K16ac è deacetilata da HDAC1,2 durante la replicazione del DNA 6. Inibisce H4K16acattività remodeler cromatina del familiare ISWI SMARCA5 12. Quindi, utilizzando la tecnica BrdU-CHIP-Slot-occidentale, potremmo collegare le funzioni per HDAC1,2 nel regolare il rimodellamento della cromatina durante la replicazione del DNA. Quindi, la tecnica BrdU-Chip-Slot-Western Blot è un approccio efficace per misurare quantitativamente l&#39;associazione e dissociazione dinamica delle proteine ​​o loro modificazioni post-traslazionali che sono tenuti al DNA nascente.

<table border="0" cellpadding="0" cellspacing="0" width="608">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-BrdU (westerns)</td>
			<td style="width:107px;">BD Biosciences</td>
			<td style="width:119px;">B555627</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-SMARCA5</td>
			<td>Abcam</td>
			<td style="width:119px;">ab3749</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-H4K16ac</td>
			<td style="width:107px;">Active Motif</td>
			<td>39167</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Zeta-Probe GT Membrane</td>
			<td style="width:107px;">Bio-Rad</td>
			<td style="width:119px;">162-0197</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Formaldehyde</td>
			<td style="width:107px;">Fisher</td>
			<td style="width:119px;">BP531-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Protein A agarose</td>
			<td style="width:107px;">Millipore</td>
			<td style="width:119px;">16-156</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rnase A</td>
			<td style="width:107px;">Qiagen</td>
			<td style="width:119px;">19101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Proteinase K</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">P4850</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PCR Purification Kit</td>
			<td style="width:107px;">Qiagen</td>
			<td style="width:119px;">28106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycine</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">G7403</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Protease inhibitor cocktail</td>
			<td style="width:107px;">Roche</td>
			<td style="width:119px;">11836170001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BrdU</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">B9285</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rabbit IgG&#160;</td>
			<td style="width:107px;">Millipore</td>
			<td style="width:119px;">12-370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">S-3014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton-X-100</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">93443</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NP40</td>
			<td style="width:107px;">USB Corporation</td>
			<td style="width:119px;">19628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium deoxycholate</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">D6750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium bicarbonate</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">S-4019</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethanol</td>
			<td style="width:107px;">Decon Laboratories</td>
			<td style="width:119px;">04-355-222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DEPC-treated water</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">95284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris</td>
			<td style="width:107px;">Fisher</td>
			<td style="width:119px;">BP-152-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:107px;">Invitrogen</td>
			<td style="width:119px;">15575-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SDS</td>
			<td style="width:107px;">Ambion</td>
			<td style="width:119px;">AM9820</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium hydroxide</td>
			<td style="width:107px;">Mallinckrodt GenAR&#160;</td>
			<td style="width:119px;">MAL7772-06</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">20X SSC</td>
			<td style="width:107px;">Life Technologies</td>
			<td style="width:119px;">15557-036</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Non-fat dry milk</td>
			<td style="width:107px;">Lab Scientific Inc</td>
			<td style="width:119px;">M0841</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ECL</td>
			<td style="width:107px;">Thermo Fisher</td>
			<td style="width:119px;">80196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">X-ray film</td>
			<td style="width:107px;">Genesee Sci</td>
			<td style="width:119px;">30-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Developer</td>
			<td style="width:107px;">Konica Minolta</td>
			<td style="width:119px;">SRX-101A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UV Crosslinker</td>
			<td style="width:107px;">Stratagene</td>
			<td style="width:119px;">XLE-1000</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Sonicator</td>
			<td style="width:107px;">Branson Sonifier&#160; Digital Ultrasonic Cell Disruptor</td>
			<td style="width:119px;">Model: 450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:107px;">Eppendorf</td>
			<td style="width:119px;">Model: 5810R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Water bath</td>
			<td style="width:107px;">Fisher Scientific Isotemp</td>
			<td style="width:119px;">Model: 2322</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NanoDrop 1000 spectrophotometer</td>
			<td style="width:107px;">ThermoScientific</td>
			<td style="width:119px;">Model: 1000</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Slot Blot apparatus&#160;</td>
			<td style="width:107px;">Schleicher and Schuell Minifold II</td>
			<td style="width:119px;">44-27570</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Tissue Culture Incubator</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:119px;">Series II 3110 Water-Jacketed CO2 Incubators</td>
			<td></td>
		</tr>
	</tbody>
</table>

examination of proteins bound to nascent dna in mammalian cells using  brdu-chip-slot-western technique

Istone deacetilasi 1 e 2 (HDAC1,2) localizzano ai siti di replicazione del DNA. Nello studio precedente, utilizzando un inibitore selettivo e di un sistema atterramento genetica, abbiamo dimostrato le funzioni nuovi per HDAC1,2 nella progressione della forcella replica e nascente manutenzione cromatina in cellule di mammifero. Inoltre, abbiamo utilizzato una tecnica BrdU-chip-Slot-occidentale che combina immunoprecipitazione della cromatina (ChIP) di bromo-deossiuridina (BrdU) DNA -labeled con macchia slot e occidentale analisi per misurare quantitativamente proteine ​​o modificazione degli istoni associati con il DNA nascente. Attivamente cellule in divisione sono state trattate con HDAC1,2 inibitore selettivo o trasfettate con siRNA contro HDAC1 e HDAC2 e quindi il DNA di nuova sintesi è stato etichettato con il bromodeossiuridina timidina analogico (BrdU). L&#39;etichettatura BrdU è stata fatta in un punto di tempo in cui non c&#39;era significativo arresto del ciclo cellulare o apoptosi a causa della perdita delle funzioni HDAC1,2. Seguendo lAbeling di cellule con BrdU, immunoprecipitazione della cromatina (ChIP) di marchi di acetilazione degli istoni o la cromatina-remodeler è stata effettuata con anticorpi specifici. BrdU marcato DNA ingresso e l&#39;immunoprecipitato (o chip) DNA è stato poi trasferite su una membrana con la tecnica di slot blot ed immobilizzati mediante UV. La quantità di DNA nascente in ciascuno slot è stata poi valutata quantitativamente mediante analisi Western con un anticorpo anti-BrdU. L&#39;effetto della perdita di funzioni HDAC1,2 sui livelli di nuova sintesi degli istoni marchi acetilazione DNA associate e cromatina remodeler è stato quindi determinato normalizzando il segnale di BrdU-Chip ottenute dai campioni trattati ai campioni di controllo.

Per determinare la specificità degli inibitori selettivi HDAC1,2, sono stati utilizzati Hdac1,2 FL / FL e HDAC3 FL / FL cellule fibrosarcoma. -Adenovirus contenenti Cre ricombinasi (Ad-Cre) è stato utilizzato per eliminare Hdac1,2 e HDAC3 in queste cellule. A seguito di infezione Ad-Cre di Hdac1,2 FL / FL cellule, è stato osservato un robusto incremento della H4K5ac. Il trattamento dell...

Watch this Scientific Journal Video about Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique at JoVE.com

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

Esame di proteine ​​legate al DNA Nascente in cellule di mammifero utilizzando la tecnica BrdU-chip-Slot-occidentale

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

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Research

JoVE Journal

Biology

9.1K Views.  University of Utah School of Medicine. In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA. 

Video: Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

A lungo termine Imaging cellule di mammifero utilizzando Wide-Field Microscopia

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Biologia

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

Transfection DNA dei mammiferi muscoli scheletrici con In Vivo Elettroporazione

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to immortalized cell lines. However, the post-mitotic nature of primary neurons prevents effective heterologous protein expression using common procedures such as electroporation or chemically-mediated transfection. Thus, other techniques must be employed in order to effectively express proteins in these non-dividing cells.
In this article, we describe the steps required to perform intranuclear injections of cDNA constructs into dissociated adult sympathetic neurons. This technique, which has been applied to different types of neurons, can successfully induce heterologous protein expression. The equipment essential for the microinjection procedure includes an inverted microscope to visualize cells, a glass injection pipet filled with cDNA solution that is connected to a N2(g) pressure delivery system, and a micromanipulator. The micromanipulator coordinates the injection motion of microinjection pipet with a brief pulse of pressurized N2 to eject cDNA solution from the pipet tip. 
This technique does not have the toxicity associated with many other transfection methods and enables multiple DNA constructs to be expressed at a consistent ratio. The low number of injected cells makes the microinjection procedure well suited for single cell studies such as electrophysiological recordings and optical imaging, but may not be ideal for biochemical assays that require a larger number of cells and higher transfection efficiencies. Although intranuclear microinjections require an investment of equipment and time, the ability to achieve high levels of heterologous protein expression in a physiologically relevant environment makes this technique a very useful tool to investigate protein function.

Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays.

Microiniezione intranucleari di DNA in dissociato neuroni dei mammiferi adulti

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to ...

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

Analisi del DNA doppio filamento Break (DSB) di riparazione in cellule di mammifero

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development.
Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope.

DT40, a model vertebrate genetic system, provides a powerful tool to analyze protein function. Here we describe a simple method that allows qualitative analysis of parameters that influence DNA synthesis during the S-phase in DT40 cells at the single molecule level.

Visualizzazione della replicazione del DNA nei vertebrati DT40 sistema modello utilizzando la tecnica del DNA Fiber

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Analisi della posizione ciclo cellulare nelle cellule di mammifero

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolamento dei ribosomi Polipeptidi Bound nascenti<em&gt; In vitro</em&gt; Per identificare i siti di pausa traslazione lungo mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Utilizzando Sequence SECM arresto come uno strumento per isolare ribosoma Polipeptidi Bound

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

In tempo reale Imaging del singolo Engineered RNA trascritti in cellule viventi Utilizzando Ratiometric bimolecolari Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

Identificazione delle proteine ​​partner di interazione in cellule di mammifero Uso SILAC-immunoprecipitazione quantitativi Proteomica

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

Esame di proteine legate al DNA Nascente in cellule di mammifero utilizzando la tecnica BrdU-chip-Slot-occidentale

Riepilogo

Esplora Altri Video

Capitoli in questo video

A lungo termine Imaging cellule di mammifero utilizzando Wide-Field Microscopia

Transfection DNA dei mammiferi muscoli scheletrici con In Vivo Elettroporazione

Microiniezione intranucleari di DNA in dissociato neuroni dei mammiferi adulti

Analisi del DNA doppio filamento Break (DSB) di riparazione in cellule di mammifero

Visualizzazione della replicazione del DNA nei vertebrati DT40 sistema modello utilizzando la tecnica del DNA Fiber

Analisi della posizione ciclo cellulare nelle cellule di mammifero

Isolamento dei ribosomi Polipeptidi Bound nascenti

Utilizzando Sequence SECM arresto come uno strumento per isolare ribosoma Polipeptidi Bound

In tempo reale Imaging del singolo Engineered RNA trascritti in cellule viventi Utilizzando Ratiometric bimolecolari Beacons

Identificazione delle proteine partner di interazione in cellule di mammifero Uso SILAC-immunoprecipitazione quantitativi Proteomica

Esame di proteine ​​legate al DNA Nascente in cellule di mammifero utilizzando la tecnica BrdU-chip-Slot-occidentale

Riepilogo

Esplora Altri Video

Capitoli in questo video

A lungo termine Imaging cellule di mammifero utilizzando Wide-Field Microscopia

Transfection DNA dei mammiferi muscoli scheletrici con In Vivo Elettroporazione

Microiniezione intranucleari di DNA in dissociato neuroni dei mammiferi adulti

Analisi del DNA doppio filamento Break (DSB) di riparazione in cellule di mammifero

Visualizzazione della replicazione del DNA nei vertebrati DT40 sistema modello utilizzando la tecnica del DNA Fiber

Analisi della posizione ciclo cellulare nelle cellule di mammifero

Isolamento dei ribosomi Polipeptidi Bound nascenti

Utilizzando Sequence SECM arresto come uno strumento per isolare ribosoma Polipeptidi Bound

In tempo reale Imaging del singolo Engineered RNA trascritti in cellule viventi Utilizzando Ratiometric bimolecolari Beacons

Identificazione delle proteine ​​partner di interazione in cellule di mammifero Uso SILAC-immunoprecipitazione quantitativi Proteomica

Esame di proteine legate al DNA Nascente in cellule di mammifero utilizzando la tecnica BrdU-chip-Slot-occidentale

Identificazione delle proteine partner di interazione in cellule di mammifero Uso SILAC-immunoprecipitazione quantitativi Proteomica