The authors thank Kerstin Glensk for preparing the MTases M.BseCI and Set7/9 and gratefully acknowledge funding by the Excellence Initiative of the German Federal and State Governments and RWTH Aachen University. The authors are happy to provide 6BAz and SeAdoYn or other cofactor analogues for collaborative research.

1. Istruzioni generali <ol><li> Conservare aziridina cofattore 6BAz (in DMSO) e di proteine ​​MTase Set7 / 9 a -80 ° C e tutti gli altri reagenti tra cui doppio attivato cofattore SeAdoYn e DNA MTase M.BseCI (nel 50% glicerolo) a -20 ° C. </li><li> Determinare la concentrazione di 6BAz e SeAdoYn tramite spettroscopia UV / Vis utilizzando i coefficienti di estinzione ε 269nm (6BAz) = 16000 centimetri -1 m -1 e ε 260nm (SeAdoYn) = 15400 centimetri -1 M -1 in acqua deionizzata. Determinare la concentrazione di MTases dal saggio Bradford o, se il coefficiente di estinzione è disponibile, tramite assorbimento diretto a 280 nm. </li><li> Cercate di evitare di creare bolle pipettando intensivo o vortex per prevenire la perdita di attività enzimatica. Invece, mescolare delicatamente pipettando su e giù. </li><li> Quando si aggiungono cofattori aziridina da soluzioni di riserva in DMSO fare in modo che la concentrazione DMSO finale nel test è menoal 5%. Includere sempre 10 ioni magnesio mm nella tampone per evitare reazioni aspecifiche con DNA. </li><li> Quando si aggiungono cofattori doppie attivati ​​da soluzioni stock acide utilizzare piccoli volumi (soluzioni stock altamente concentrati) per evitare variazioni di pH e di fare in modo che il pH della soluzione test non cambia in modo significativo. Evitare tioli, ad esempio, β-mercaptoetanolo o ditiotreitolo (DTT), in tampone perché possono interferire con la reazione di scatto per complessazione degli ioni rame richiesti. </li></ol> 2. One-step Etichettatura del DNA tramite aziridina Cofattori <ol><li> Metiltransferasi-Induced Label ing specifici Sequence (sorridendo) di plasmide DNA con M.BseCI MTase DNA e aziridina cofattore 6BAz. <ol><li> Scongelare la soluzione cofattore a 20 ° C e preparare le miscele di reazione in ghiaccio. </li><li> Oltre al dosaggio effettuare un controllo &quot;cofattore&quot;, di visualizzare le modifiche specifiche non e un &amp; #8220; enzima &quot;controllo, per assicurarsi che la preparazione MTase è libero di naturale AdoMet cofattore. </li><li> Per il mix dosaggio di 2 ml di 10x tampone di modifica (contenente 100 mM Tris-HCl, 100 mM MgCl 2, 20 mM β-mercaptoetanolo, pH 7,4), 2 ml di pBR322 (0,5 mg / mL), 10 eq. M.BseCI per sequenza di riconoscimento sul DNA (sequenza 1 riconoscimento in pBR322) e il cofattore aziridina 6BAz ad una concentrazione finale di 60 mM in un volume totale di 20 microlitri. Aggiungere cofattore e MTase DNA scorso. NOTE: β-Mercaptoethanol è tossico, corrosivo e dannosi per l&#39;ambiente. </li><li> Per il controllo &quot;cofattore&quot; aggiungere acqua deionizzata invece di M.BseCI e per il controllo &quot;enzima&quot; aggiungere acqua deionizzata al posto di 6BAz. </li><li> Mescolare le soluzioni pipettando gentilmente su e giù. </li><li> Incubare le provette a 55 ° C per 1 ora. </li><li> Centrifugare brevemente per raccogliere tutto il liquido sul fondo delle provette. </li></ol></li><li> Test restrizione-modificazione per verificare la modifica del DNA. <ol><li> Preparare una soluzione mescolando 10 microlitri tampone 10x R.TaqI (contenente 100 mM Tris-HCl, 50 mM MgCl 2, 1 M NaCl, 1 mg / ml di albumina di siero bovino, pH 8,0), 80 microlitri di acqua deionizzata e 3,3 ml di endonucleasi di restrizione (REase) da Thermus aquaticus (R.TaqI, 10 U / ml). Assicurarsi di aggiungere il REase nell&#39;ultimo passaggio. </li><li> Per ogni tubo da 2.1.7 aggiungere 2 ml di 10x tampone R. TaqI e 28 ml di soluzione dall&#39;alto (2.2.1). </li><li> Mescolare le soluzioni pipettando gentilmente su e giù. </li><li> Incubare le provette a 65 ° C per 30 min. </li><li> Centrifugare brevemente per raccogliere tutto il liquido sul fondo delle provette. </li></ol></li><li> Assay spostamento elettromobilità (EMSA) con streptavidina per verificare la modifica funzionale. <ol><li> Rimuovere 25 microlitri da ogni provetta (2.2.5) e aggiungere 2,4 ml di una soluzione di streptavidina (1 mM rispetto alla streptavidina monomer in tampone contenente streptavidina 100 mM Na 2 HPO 4, NaCl 100 mM, pH 7,5; 4 equivalenti di biotina totale). Aggiungere 2,4 ml di tampone streptavidina ai rimanenti tubi. </li><li> Incubare tutte le provette a 37 ° C per 1 ora. </li></ol></li><li> Analisi tramite elettroforesi su gel di agarosio. <ol><li> Aggiungere 5 ml di tampone di caricamento 6x (0,25% blu di bromofenolo, 30% glicerolo) ad ogni provetta. </li><li> Mescolare le soluzioni delicatamente. </li><li> Carico 10 microlitri di ciascun campione nei pozzetti di un gel di agarosio (1% agarosio in tampone TBE 0,5x contenente 1x GelRed da una soluzione 10.000X magazzino). </li><li> Eseguire il gel in tampone TBE 0.5x con 80 V per circa. 1 ora. </li><li> Visualizzare bande di DNA su un tavolo UV (312 nm) con una telecamera CCD dotata di filtro (540 ± 50 nm). NOTA: la luce UV è dannoso per gli occhi e la pelle. </li></ol></li></ol> 3. Due-passo Protein Labeling via Cofattori doppie Attivato <ol><li> MethyltransfTrasferimento di gruppi attivati ​​(MTAG) con Set7 / 9 e doppio attivato cofattore SeAdoYn per dell&#39;istone H3 lisina 4 Etichettatura (fase di modifica) cancella-Diretto. <ol><li> Scongelare i componenti e preparare le miscele di reazione sul ghiaccio. NOTA: Tenere sempre SeAdoYn raffreddato per evitare il degrado. </li><li> Oltre al dosaggio effettuare un controllo &quot;cofattore&quot;, di visualizzare le modifiche non specifiche, e un controllo &quot;enzima&quot;, di escludere reazioni non specifiche della sonda fluorescente. </li><li> Preparare una soluzione test (20 microlitri) contenente tampone modifica (50 mM Tris-HCl, 5% glicerolo, pH 8,5), 10 micron istone H3, 10 micron Set7 / 9 e 600 micron SeAdoYn (miscela di entrambi epimeri al selenio). Negli ultimi passi aggiungere cofattore e poi MTase. </li><li> Per il controllo &quot;cofattore&quot; preparare una soluzione test come in 3.1.3 e aggiungere 60 AdoMet mM di competere con il cofattore sintetico. Per il controllo &quot;enzima&quot; aggiungere acqua deionizzata al posto di SeAdoYn.</li><li> Mescolare le soluzioni lentamente pipettando su e giù. Controllare il pH aggiungendo 1 ml di ciascuna soluzione di campo superiore di una striscia pH (range pH di 5 - 10). </li><li> Incubare a 37 ° C per 2 ore. </li><li> Nel frattempo preparare un gel SDS gel di poliacrilammide al 12% (in esecuzione: 357 Bis-Tris pH 6,5-6,8 mM, 0,1% (w / v) APS, 0,04% (v / v) TEMED e 12% acrilammide / bisacrilammide 37.5: 1 ; loading gel: 357 Bis-Tris pH 6,5-6,8 mM, 0,1% (w / v) APS, 0,04% (v / v) TEMED e 5% acrilammide / bisacrilammide 37,5: 1). NOTA: L&#39;acrilamide / bisacrilammide è tossico e la salute pericolosi. Indossare guanti durante questa procedura. </li></ol></li><li> Etichettatura chimica di lisina alkinylated 4 in H3 via azide-alkyne cicloaddizione (CuAAC) (step etichettatura) di rame-catalizzata. <ol><li> Poco prima della fine della reazione modifica preparare una miscela contenente 3 5x click CuSO 4 mM, tris 3 mM (3-idrossipropil-triazolylmethyl) ammina (THPTA), 250 mM ascorbato di sodio e 6 mM TAMRA azide convolume totale di 20 microlitri. </li><li> Aggiungere 5 ml di 5x click mix preparato fresco ogni provetta per avviare il CuAAC e placare la reazione di modifica. </li><li> Mescolare delicatamente pipettando su e giù. </li><li> Proteggere tutti i tubi con un foglio di alluminio dalla luce per evitare fotometabolismo del fluoroforo. </li><li> Incubare a 20 ° C per 1 ora. </li></ol></li><li> Protein precipitazioni per rimuovere l&#39;eccesso di libera TAMRA fluoroforo. <ol><li> Per evitare eclissare della fluorescenza etichettati istone H3 da intensa fluorescenza in gel di libera TAMRA fluoroforo, eliminare l&#39;eccesso di fluoroforo per precipitazione delle proteine ​​(3.3.2 - 3.3.4) 34. </li><li> Aggiungere 75 ml di metanolo, 18,8 ml cloroformio e 50 ml di acqua deionizzata ad ogni provetta e vortex brevemente dopo ogni aggiunta. Centrifugare a 16.000 g per 5 min. Rimuovere la fase superiore senza disturbare lo strato di interfaccia, che contiene la proteina. </li><li> Aggiungere 56,3 ml di metanolo al rimanente fase in ogni provetta, vortex e centrifugare a 16.000 xg per 5 min a pellet proteina. Rimuovere il surnatante. Ripetere questo passaggio per lavare il pellet. </li><li> Coprire i tubi aperti con un panno che non lasci residui e lasciare asciugare per 15-30 min. </li></ol></li><li> Analisi tramite SDS PAGE. <ol><li> Sciogliere proteine ​​precipitate da 3.3.4 in 20 microlitri tampone di SDS carico (50 mM Tris-HCl, 2,5% (w / v) di SDS, 10% (v / v) di glicerolo, 320 mM β-mercaptoetanolo e 0,05% (w / v) blu bromofenolo, pH 6,8). Assicurati di sciogliere completamente il pellet sciacquando le pareti dei tubi con una pipetta. </li><li> Incubare i campioni a 95 ° C per 10 minuti e lasciate raffreddare a 20 ° C. </li><li> Centrifugare brevemente per raccogliere tutto il liquido sul fondo delle provette. </li><li> Caricare l&#39;intero importo di ogni campione nei pozzetti di un gel di poliacrilammide SDS (3.1.7). Utilizzare 50 MOPS mM, 50 mM Tris-X (Tris-base), 5 mM EDTA, 0,1% (w / v) di SDS come tampone di corsa per elettroforesi. </li><li>Eseguire il gel con 120 V per circa. 90 min. </li><li> Visualizzare la fluorescenza in-gel su un tavolo UV (312 nm) con una telecamera CCD dotata di filtro (540 nm ± 50 nm). NOTA: la luce UV è dannoso per gli occhi e la pelle. </li></ol></li></ol>

The authors disclose the following competing financial interest: E.W. is inventor on related patents.

Etichettatura in un unico passaggio di DNA con MTases DNA e cofattori aziridina (DNA sorridendo) è un metodo robusto ma alcuni aspetti deve essere considerato al momento di pianificare l&#39;esperimento. Cofattore Aziridine: La concentrazione 6BAz per l&#39;etichettatura del DNA con M.BseCI era di 60 micron. Quando si utilizzano altri MTases DNA la concentrazione cofattore dovrebbe essere ottimizzato, ad esempio concentrazioni partire da 20 micron sono stati impiegati con ...

Etichettatura specifica degli acidi nucleici e proteine ​​1,2 3,4 è di grande interesse per le caratterizzazioni funzionali, diagnosi medica e (nano) biotecnologie. Qui vi presentiamo un metodo enzimatico di etichettatura per questi biopolimeri che si basa su S -adenosyl-L-metionina (SAM AdoMet o) metiltransferasi-dipendente (MTases). Questa classe di enzimi (EC 2.1.1.) Rivolge singole posizioni nucleofili (azoto, ossigeno, zolfo e atomi di carbonio) all&#39;interno specifici residui di acidi nucleici e proteine ​​e naturalmente trasferisce il gruppo metilico attivato della AdoMet cofattore (Figura 1A) 5. Inoltre, MTases possono utilizzare analoghi cofattore sintetici per l&#39;etichettatura specifico con tag affinità, fluorofori o altre etichette (Figura 1B) 6. Sono stati sviluppati due classi di analoghi AdoMet: cofattori Aziridine per S equence-specifica M ethyltransferase- I </strong&gt; Nduced L abel ing (sorridendo) 7 e doppie analoghi attivati ​​AdoMet per m ethyltransferase diretto T rasferimento di un ruppi G ctivated (MTAG) 8. <img alt="Figura 1" fo:content-width="7in" src="/files/ftp_upload/52014/52014fig1highres.jpg" width="700" /> Figura 1: reazioni catalizzate da metiltransferasi (MTases) A. metile trasferimento gruppo dal cofattore naturale AdoMet (SAM) per vari substrati, tra cui il DNA, RNA, proteine ​​e piccole biomolecole B. Etichettatura / funzionalizzazione di acidi nucleici e proteine ​​(NNNNN =.. coppie di basi di DNA, RNA nucleotidi per e aminoacidi delle proteine; XXXXX = sequenza di riconoscimento del MTase con residuo di destinazione in verde) con analoghi cofattore sintetici. Cofattori aziridina contenenti un gruppo giornalista (sfera blu)collegata all&#39;anello di adenina sono sequenza specificamente accoppiato con il residuo di destinazione (a sinistra) e analoghi doppio attivato AdoMet comportare un trasferimento del catene alchiliche estese trasportano un giornalista chimica Y (a destra), che può essere etichettato da bioorthogonal reazione scatto in una seconda fase. <a href="https://www.jove.com/files/ftp_upload/52014/52014fig1highres.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> Cofattori aziridina funzionano meglio con DNA MTases. Essi contengono un anello di tre termini con un atomo di azoto 9 (o un N -mustard 10,11) invece del centro solfonio gruppo reattivo. Protonazione di questo atomo di azoto attiva il azirdinico per l&#39;attacco nucleofilo dal nucleotide obiettivo che porta ad accoppiamento covalente di tutta cofattore con DNA. Collegando gruppi giornalista all&#39;anello adenina cofattori aziridina possono essere utilizzati in combinazione con DNA MTases etichettare DNA in un solo passo ( <stron<html> g&gt; Figura 1B, a sinistra) 7,12. Ciò è illustrato nel dettaglio per la biotinilazione di DNA con 6BAz 13 - 15 (cofattore aziridina con biotina attaccato alla posizione 6 dell&#39;anello adenina) e il DNA MTase specifico adenina da Bacillus stearothermophilus (M.BseCI) 16 (figura 2, vedi sezione Protocollo 2: etichettatura One-step del DNA tramite aziridina cofattori). Oltre a M.BseCI (&#39;sequenza di riconoscimento, il MTases DNA da Thermus aquaticus (M.TaqI, 5&#39;-TCG A -3 5&#39;-ATCG A T-3)&#39;), da Haemophilus heamolyticus (M.HhaI, 5 &#39;-G C GC-3&#39;) e da Spiroplasma (M.SssI, 5&#39;-C G-3 &#39;) sono stati utilizzati con successo per biotinilato DNA con 6BAz 17. Inoltre, cofattori aziridina possono essere impiegati per un passo etichettatura fluorescenza DNA 18,19. ONTENUTO &quot;fo: keep-together.within-page =&quot; always &quot;&gt; <img alt="Figura 2" fo:content-width="7in" src="/files/ftp_upload/52014/52014fig2highres.jpg" width="700" /> Figura 2:. Sequenza specifica biotinilazione un passo del DNA con M.BseCI e 6BAz Il DNA MTase M.BseCI riconosce la doppia elica sequenza di DNA 5&#39;-ATCG A T-3 &#39;e naturalmente metila il gruppo amminico del secondo adenina residuo (verde) con AdoMet. Con il cofattore aziridinico 6BAz il corso della reazione viene modificato e M.BseCI porta alla specifica sequenza biotinilazione DNA accoppiando tutta cofattore inclusi biotina (blu) con l&#39;adenina destinazione. <a href="https://www.jove.com/files/ftp_upload/52014/52014fig2highres.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> Doppia analoghi AdoMet attivati ​​contiene estese catene laterali insature invece di un gruppo metile al centro solfonio (Figura 1B </strong&gt;, a destra) 20. Il legame doppio o triplo insaturo in β-position al centro solfonio compensa elettronicamente effetti sterici sfavorevoli all&#39;interno dello stato di transizione di stabilizzazione coniugativo. Dal momento che sia il centro solfonio e il legame insaturo attivare la catena laterale per il trasferimento enzimatico, questi cofattori sono stati nominati analoghi AdoMet doppio attivati. In genere, essi sono utilizzati per trasferire catene laterali con gruppi unici chimici (giornalisti chimici), come gruppi di aminoacidi, alchini e azide, per l&#39;etichettatura chemio-selettivo in una seconda fase 8,21. In generale, analoghi a doppia attivato AdoMet non può funzionare solo come cofattori per DNA MTases 8,20,21 ma anche per RNA MTases 22,23 e MTases proteine ​​24-28 permettendo etichettatura supplementare di RNA e proteine. Tuttavia, le catene laterali estese sono stericamente più esigenti di un gruppo metile e allargando le MTase siti attivi di proteine ​​di ingegneria è often necessaria per ottenere velocità di trasferimento efficienti. Un&#39;altra soluzione a questo problema è di utilizzare un analogo AdoMet con un piccolo gruppo propargile (tre atomi di carbonio) in cui l&#39;alchino terminale ha due funzioni: 1. Stabilizzazione dello stato di transizione durante il trasferimento enzimatica e 2. maniglia reattiva per seguenti modificazioni chimiche di rame catalizzata cicloaddizione azide-alchino (CuAAC) clicca chimica. Si è scoperto che il propargilico risultante AdoMet analogico 29 è abbastanza instabile in condizioni di neutralità o leggermente di base e di scarsa utilità. Questo inconveniente può essere risolto sostituendo l&#39;atomo di zolfo con selenio. Il cofattore risultante 5 &#39;- [(Se) [(3 S) -3-ammino-3-carbossipropil] prop-2-ynylselenonio] -5&#39;-deossiadenosina (SeAdoYn, figura 3) è accettata da wild-type DNA, RNA e MTases proteine ​​30-32 che abolisce la necessità di ingegneria proteica in molti casi. Questo è esemplificato da fluorescenza pro<html> etichettatura proteina con la lisina istone H3 4 (H3K4) MTase Set7 / 9 33 (Figura 3, vedere la sezione protocollo 3: etichettatura proteina Two-step con cofattori doppie attivati). <img alt="Figura 3" fo:content-width="7in" src="/files/ftp_upload/52014/52014fig3highres.jpg" width="700" /> Figura 3:. In due fasi un&#39;etichettatura specifica fluorescenza-Sequenza dell&#39;istone H3 con Set7 / 9, SeAdoYn e TAMRA azide La proteina MTase Set7 / 9 metila naturalmente il gruppo amminico di lisina 4 a dell&#39;istone H3 (H3K4, verde) con AdoMet. Con il cofattore doppio attivato SeAdoYn il MTase trasferisce un piccolo gruppo propargile (rosso) per il residuo di lisina. Il legame terminale collegato tripla è poi selettivamente modificata in una reazione click bioorthogonal (azide-alkyne cicloaddizione di rame-catalizzata, CuAAC) con azide-derivatizzata TAMRA (tetrametilrodamina, blu) fluoroforo.Carico / 52014 / 52014fig3highres.jpg &quot;target =&quot; _ blank &quot;&gt; Clicca qui per vedere una versione più grande di questa figura.

<table border="0" cellpadding="0" cellspacing="0" width="669">
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			<td height="21" style="height:21px;width:245px;">Name of Reagent/ Equipment</td>
			<td style="width:139px;">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;width:245px;">6BAz</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Synthesized according to Weinhold et al., Patent number US 8,129,106, published March 6, 2012.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">&#946;-Mercaptoethanol</td>
			<td style="width:139px;">Serva</td>
			<td align="right" style="width:119px;">28625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Acetic acid</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td align="right" style="width:119px;">10304980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Acrylamide/Bis Solution, 37.5:1</td>
			<td style="width:139px;">Serva</td>
			<td align="right" style="width:119px;">10688</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">UltraPure Agarose</td>
			<td style="width:139px;">Invitrogen</td>
			<td align="right" style="width:119px;">16500100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Ammonium persulfate (APS)</td>
			<td style="width:139px;">Serva</td>
			<td align="right" style="width:119px;">13375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Bis-Tris</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1304</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Boric acid</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Bromophenol blue Na salt</td>
			<td style="width:139px;">Serva</td>
			<td align="right">15375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Copper(II) sulfate</td>
			<td style="width:139px;">Aldrich</td>
			<td style="width:119px;">C1297</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Chloroform</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td align="right">10020090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Coomassie Brilliant Blue</td>
			<td style="width:139px;">Serva</td>
			<td align="right" style="width:119px;">17525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">EDTA disodium salt</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Ethanol</td>
			<td style="width:139px;">Merck</td>
			<td align="right" style="width:119px;">100983</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">GelRed (10000x in water)</td>
			<td style="width:139px;">Biotium</td>
			<td align="right" style="width:119px;">41003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Glycerol (99.5%)</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">2006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">FastRuler Low Range DNA Ladder</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td style="width:119px;">SM1103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Histone H3</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Expressionplasmid obtained from Dr. Philipp Voigt and Prof. Danny Reinberg; expression and isolation according to T. J. Richmond et al., J. Mol. Biol. 1997, 272, 301-311.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">M.BseCI</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Expressionplasmid obtained from Dr. Michael Kokkinidis; expression and isolation according to Kapetaniou et al., Acta Cryst. 2006, F63, 12-14.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Methanol</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td align="right" style="width:119px;">10675112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Magnesiumchloride&#160; hexahydrate</td>
			<td style="width:139px;">J.T. Baker</td>
			<td align="right" style="width:119px;">4003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">MOPS</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Sodium chloride</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">pH strip (Neutralit)</td>
			<td style="width:139px;">Merck</td>
			<td align="right" style="width:119px;">1,095,330,001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">pBR322</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td style="width:119px;">SD0041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">R.TaqI (10u/&#181;l)</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td>ER0671</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">SeAdoYn</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Synthesized according to Willnow et al., ChemBioChem 2012, 13, 1167-1173.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Set7/9</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Expressionplasmid obtained from Prof. Danny Reinberg, expression and isolation according to D. Reinberg et al., Genes Dev.2002, 16, 479-489.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Streptavidin</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">3058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">(+)-Sodium L-ascorbate</td>
			<td style="width:139px;">Sigma Life Science</td>
			<td style="width:119px;">A7631</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">SDS Granular</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1833</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">di-Sodiumhydrogenphosphat</td>
			<td style="width:139px;">Merck</td>
			<td align="right" style="width:119px;">106,586</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">TAMRA-azide</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">TaqI buffer (10x)</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td style="width:119px;">B28</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:245px;">N,N,N&#39;,N&#39;-Tetramethylethylenediamine (TEMED)</td>
			<td style="width:139px;">Acros Organics</td>
			<td align="right" style="width:119px;">42058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Tris-HCl</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Tris-X (TRIS-base)</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1018</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">Tris(3-hydroxypropyltriazolyl-methyl)amine (THPTA)</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">762342</td>
			<td></td>
		</tr>
	</tbody>
</table>

sequence-specific labeling of nucleic acids and proteins with methyltransferases and cofactor analogues

S -Adenosyl-L-metionina (SAM) o AdoMet metiltransferasi-dipendente (MTase) catalizzano il trasferimento del gruppo metilico attivato dal AdoMet a specifiche posizioni in DNA, RNA, proteine ​​e piccole biomolecole. Questa reazione di metilazione naturale può essere espansa a un&#39;ampia varietà di reazioni di alchilazione con analoghi sintetici cofattore. Sostituzione del centro solfonio reattiva di AdoMet con un anello di aziridina conduce cofattori accoppiabili con DNA da vari MTases DNA. Questi cofattori aziridina possono essere equipaggiati con gruppi reporter diverse posizioni della frazione adenina e utilizzati per S-specific equence M ethyltransferase- I nduced L Abel zione del DNA (sorridendo DNA). Come esempio tipico diamo un protocollo per biotinilazione di pBR322 plasmide DNA sequenza 5&#39;-ATCG A T-3 &#39;con il DNA MTase M.BseCI e il cofattore aziridina 6BAz inun passo. Estensione del gruppo metilico attivato con insaturi gruppi alchil risultati in un&#39;altra classe di analoghi AdoMet che sono utilizzati per m ethyltransferase diretto T rasferimento di un ctivated ruppi G (MTAG). Poiché le catene laterali estesi vengono attivati ​​dal centro solfonio e il legame insaturo, questi cofattori sono chiamati analoghi AdoMet doppio attivati. Questi analoghi funzione non solo come cofattori per DNA MTases, come cofattori aziridina, ma anche per RNA, proteine ​​e piccole molecole MTases. Essi sono tipicamente utilizzati per modificazione enzimatica di substrati MTase con gruppi funzionali uniche che sono contrassegnati con gruppi del reporter in una seconda fase chimica. Questo è esemplificato in un protocollo per l&#39;etichettatura di fluorescenza di proteine ​​istone H3. Un piccolo gruppo propargile viene trasferito dal SeAdoYn cofattore analogica alla proteina dalla lisina istone H3 4 (H3K4) MTase Set7 / 9 seguito da etichettatura clic delalkynylated H3 istone con TAMRA azide. Etichettatura MTase-mediata con analoghi cofattore è una tecnologia abilitante per molte applicazioni interessanti, tra cui l&#39;identificazione e lo studio funzionale di substrati MTase e genotipizzazione del DNA e rilevazione metilazione.

One-step Etichettatura del DNA tramite aziridina Cofattori Questa reazione viene effettuata ad esempio con il DNA MTase M.BseCI, che modifica il secondo residuo di adenina nel doppio filamento 5&#39;-ATCG A T-3 &#39;sequenza ed ha un sito di riconoscimento sul plasmide pBR322 (Figura 4A). Per verificare l&#39;etichettatura plasmide, pBR322 è sfidato con la endonucleasi di restrizione (REase) R.TaqI (5&#39;-TCGA-3 &#39;). R.TaqI ha sette siti pBR322...

Watch this Scientific Journal Video about Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues at JoVE.com

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

DNA e proteine ​​sono la sequenza-specifica etichettati con affinità o gruppi reporter fluorescente con DNA o proteine ​​metiltransferasi e analoghi cofattore sintetici. A seconda della specificità cofattore di enzimi, aziridina o doppi analoghi cofattore attivati ​​sono impiegati per l&#39;etichettatura di uno o due step.

Specifici Sequenza Etichettatura degli acidi nucleici e proteine ​​con metiltransferasi e cofattore Analoghi

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

sequence-specific-labeling-nucleic-acids-proteins-with

Research

JoVE Journal

Biology

13.9K Views.  RWTH Aachen University.  DNA e proteine ​​sono la sequenza-specifica etichettati con affinità o gruppi reporter fluorescente con DNA o proteine ​​metiltransferasi e analoghi cofattore sintetici. A seconda della specificità cofattore di enzimi, aziridina o doppi analoghi cofattore attivati ​​sono impiegati per l'etichettatura di uno o due step. 

Video: Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are analyzed. Established methods to detect cell division include direct visualization by continuous microscopy in cell culture, dilution of vital dyes such as carboxy&#64258;uorescein di-aetate succinimidyl ester (CFSE), immuno-detection of mitogenic antigens such as ki67 or PCNA, and thymidine analogues. Thymidine analogues can be detected by a variety of methods including radio-detection for tritiated thymidine, immuno-detection for bromo-deoxyuridine (BrdU), chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU), and chemical detection for ethinyl-deoxyuridine (EdU). We have derived a strategy to detect sequential incorporation of different thymidine analogues (CldU and IdU) into tissues of adult mice. Our method allows investigators to accurately quantify two successive rounds of cell division. By optimizing immunostaining protocols our approach can detect very low dose thymidine analogues administered via the drinking water, safe to administer to mice for prolonged periods of time. Consequently, our technique can be used to detect cell turnover in very long-lived tissues. Optimal immunofluoresent staining results can be achieved in multiple tissue types, including pancreas, skin, gut, liver, adrenal, testis, ovary, thyroid, lymph node, and brain. We have also applied this technique to identify oncogenic transformation within tissues. We have further applied this technique to determine if transit-amplifying cells contribute to growth or renewal of tissues. In this sense, sequential administration of thymidine analogues represents a novel approach for studying the origins and survival of cells involved in tissue homeostasis.

Immunofluorescent Detection of Two Thymidine Analogues CldU and IdU in Primary Tissue

We have derived a strategy to detect sequential incorporation of thymidine analogues (CldU and IdU) into tissues of adult mice to quantify two successive rounds of cell division. This strategy is useful to detect cell turnover of long-lived tissues, oncogenic transformation, or transit-amplifying cells.

Rilevazione immunofluorescenza di due analoghi della timidina (CldU e IDU) in tessuto primario

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are ...

Ricerca

Biologia

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

Profilatura di metiltransferasi e altri S-Adenosil- L-Omocisteina-binding proteine ​​mediante spettrometria di massa Compound Capture (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

NanoDrop microvolumi quantificazione degli acidi nucleici

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

Esaminando la dinamica conformazionale delle proteine ​​di membrana In situ Con il sito-diretta Etichettatura fluorescenza

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

Etichettatura stirpe di cellule Zebrafish con laser Uncagable Fluoresceina Destrano

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF &middot; (OD260-OD320)&middot; NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1.
Protein concentration results can be expressed as &mu;g/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.

Determinazione concentrazione di acidi nucleici e proteine ​​Utilizzando il Micro-volume Bio-spec Spettrofotometro Nano

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells. While fluorescent 
proteins such as green fluorescence protein (GFP) have been extensively used as fusion partners to proteins to track the properties of a protein of interest1, recent 
developments with smaller tags enable new functionalities of proteins to be examined in cells such as conformational change and protein-association 2, 3. One small 
tag system involves a tetracysteine motif (CCXXCC) genetically inserted into a target protein, which binds to biarsenical dyes, ReAsH (red fluorescent) and FlAsH 
(green fluorescent), with high specificity even in live cells 2. The TC/biarsenical dye system offers far less steric constraints to the host protein than 
fluorescent proteins which has enabled several new approaches to measure conformational change and protein-protein interactions 4-7. We recently developed 
a novel application of TC tags as sensors of oligomerization in cells expressing mutant huntingtin, which when mutated aggregates in neurons in Huntington 
disease 7. Huntingtin was tagged with two fluorescent dyes, one a fluorescent protein to track protein location, and the second a TC tag which only 
binds biarsenical dyes in monomers. Hence, changes in colocalization between protein and biarsenical dye reactivity enabled submicroscopic oligomer 
content to be spatially mapped within cells. Here, we describe how to label TC-tagged proteins fused to a fluorescent protein (Cherry, GFP or CFP) 
with FlAsH or ReAsH in live mammalian cells and how to quantify the two color fluorescence (Cherry/FlAsH, CFP/FlAsH or GFP/ReAsH combinations).

The biarsenical dyes FlAsH and ReAsH bind specifically to tetracysteine motifs in proteins and can selectively label proteins in live cells. Recently this labeling strategy has been used to develop sensors for different protein conformations or oligomeric states. We describe the labeling approach and methods to quantitatively analyze binding.

ReAsH / Flash Etichettatura e Image Analysis di proteine ​​sensore Tetracysteine ​​nelle celle

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells.  While fluorescent 
proteins ...

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine cell central metabolism and discover new enzymes is 13C-assisted metabolism analysis 1. This technique is based on isotopic labeling, whereby microbes are fed with a 13C labeled substrates. By tracing the atom transition paths between metabolites in the biochemical network, we can determine functional pathways and discover new enzymes. 
As a complementary method to transcriptomics and proteomics, approaches for isotopomer-assisted analysis of metabolic pathways contain three major steps 2. First, we grow cells with 13C labeled substrates. In this step, the composition of the medium and the selection of labeled substrates are two key factors. To avoid measurement noises from non-labeled carbon in nutrient supplements, a minimal medium with a sole carbon source is required. Further, the choice of a labeled substrate is based on how effectively it will elucidate the pathway being analyzed. Because novel enzymes often involve different reaction stereochemistry or intermediate products, in general, singly labeled carbon substrates are more informative for detection of novel pathways than uniformly labeled ones for detection of novel pathways3, 4. Second, we analyze amino acid labeling patterns using GC-MS. Amino acids are abundant in protein and thus can be obtained from biomass hydrolysis. Amino acids can be derivatized by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) before GC separation. TBDMS derivatized amino acids can be fragmented by MS and result in different arrays of fragments. Based on the mass to charge (m/z) ratio of fragmented and unfragmented amino acids, we can deduce the possible labeled patterns of the central metabolites that are precursors of the amino acids. Third, we trace 13C carbon transitions in the proposed pathways and, based on the isotopomer data, confirm whether these pathways are active 2. Measurement of amino acids provides isotopic labeling information about eight crucial precursor metabolites in the central metabolism. These metabolic key nodes can reflect the functions of associated central pathways.
13C-assisted metabolism analysis via proteinogenic amino acids can be widely used for functional characterization of poorly-characterized microbial metabolism1. In this protocol, we will use Cyanothece 51142 as the model strain to demonstrate the use of labeled carbon substrates for discovering new enzymatic functions.

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

13C-isotope labeling is a useful technique for determining the cell central metabolism for various types of microorganisms. After cells have been cultured with a specific labeled substrate, GC-MS measurement can reveal functional metabolic pathways based on unique labeling patterns in proteinogenic amino acids.

Percorso di conferma metaboliche e scoperta attraverso 13 C-etichettatura dei proteinogenici Aminoacidi

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

Cella di misura Kinetics progressione del ciclo con la marcatura metabolica e citometria a flusso

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Analyzing and Building Nucleic Acid Structures with 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic acid structure, including the assignment of base pairs and the determination of rigid-body parameters that describe the structure and, in Protocol 3, by a description of the reconstruction of an atomic model of a structure from its rigid-body parameters. The most recent version of 3DNA, version 2.1, has new features for the analysis and manipulation of ensembles of structures, such as those deduced from nuclear magnetic resonance (NMR) measurements and molecular dynamic (MD) simulations; these features are presented in Protocols 4 and 5. In addition to the 3DNA stand-alone software package, the w3DNA web server, located at <a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.edu</a>, provides a user-friendly interface to selected features of the software. Protocol 6 demonstrates a novel feature of the site for building models of long DNA molecules decorated with bound proteins at user-specified locations.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.

Analisi e costruzione di strutture acidi nucleici con 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

Specifici Sequenza Etichettatura degli acidi nucleici e proteine ​​con metiltransferasi e cofattore Analoghi

Specifici Sequenza Etichettatura degli acidi nucleici e proteine con metiltransferasi e cofattore Analoghi