Siamo grati a Michela de Martino, Wilfred R. Hagen, e Kourosh Honarmand Ebrahimi per utili discussioni. Questo lavoro è stato sostenuto da finanziamenti di avviamento dalla Delft University of Technology.

1. Dps espressione e purificazione Ottenimento di proteine ​​di elevata purezza è un primo passo essenziale per il saggio di DNA-protezione. Purificazione di proteine ​​Dps può essere eseguita in 4 o 5 giorni. <ol><li> Trasformare un ceppo proteasi carente di E. coli (come BL21 (DE3) pLysS) con un vettore pET (come pET17) in cui è stato clonato la sequenza codificante proteina-PS. </li><li> Streak l&#39;trasformato cellule affaccia Luria Broth (LB) piastre di agar contenenti antibiotici appropriati (come l&#39;ampicillina e cloramfenicolo per gli esempi forniti in 1.1). Incubare le piastre notte a 37 ° C. </li><li> Inoculare una singola colonia dalla piastra in 30 ml di terreno LB contenente antibiotici appropriati. Incubare per una notte a 37 ° C agitando a 200-250 rpm. </li><li> Inoculare 2 x 1 L terreno LB contenente antibiotici appropriati con 10 ml ciascuna di coltura durante la notte. Crescere a 37 ° C, agitando, a OD 600 </sub&gt; di circa 0,6. (Crescente celle a maggiori aumenti OD resa di proteine, senza compromettere sensibilmente la purezza del prodotto finale.) Indurre l&#39;espressione di Dps aggiungendo IPTG ad una concentrazione di 0,3 mM, e incubare a 37 ° C per 3-4 ore agitando. </li><li> Celle di raccolta per centrifugazione a 6000 xg per 15 min. Risospendere il pellet di cellule in 7,5 ml di DEAE tampone A (50 mM HEPES-KOH, pH 7.5, 100 mM NaCl, 0,1 mM EDTA) per L di coltura cellulare indotta. Campioni possono essere congelati in azoto liquido e conservati a -80 ° C, se desiderato. La procedura può essere continuato dalle scongelamento dei campioni in un bagno di acqua a 4 ° C. </li><li> Aggiungere una miscela di inibitori delle proteasi (come Calbiochem III set inibitori della proteasi, a 0,167 ml per ml di sospensione cellulare) alla sospensione cellulare impedire il degrado del Dps overexpressed. </li><li> Preparare l&#39;estratto privo di cellule utilizzando una stampa francese. Primo la stampa francese con 20 ml DEAE tampone A (se si utilizza un modello a flusso continuo), quindi alcuna interruzione,t il campione due volte a 20 kpsi. Centrifugare il lisato a 30.000 xg per 35 min a 4 ° C per chiarire particelle insolubili, e salvare il surnatante. Il supernatante può allora essere congelato con azoto liquido e conservato a -80 ° C, se desiderato. </li><li> Equilibrare un ml DEAE Sepharose colonna 30 CL-6B con DEAE tampone A, utilizzando un FPLC. Esegui tampone attraverso la colonna a 1-2 ml / min con una pressione massima di 0,2 MPa su sfondo. Caricare l&#39;estratto cellulare sulla colonna, e cominciare a raccogliere il flusso attraverso una volta che il segnale OD 280 comincia ad aumentare sopra del basale. Lavare la colonna con DEAE tampone A fino a quando il valore di OD 280 ritorna al basale, mentre continua la raccolta del flusso passante. Il passaggio di flusso dovrebbe contenere la maggior parte della proteina Dps, libera da DNA legato. Lavare la colonna con il 100% tampone B (50 mM HEPES-KOH, pH 7,5, 1 M di NaCl, 0,1 mM EDTA). Questo eluato contiene complessi Dps-DNA così come altre proteine ​​e può essere scartato. </li><li> Rimuovere annunciozionale proteine ​​contaminanti attraverso ammonio solfato di precipitazione. Determinare il volume del pool di flusso continuo. Lentamente (in un periodo di 10-20 min) aggiungere secco solfato di ammonio a paglia al 62% di saturazione (390 mg per ml di soluzione) a 4 ° C agitando bene. Lavorare l&#39;impasto per un ulteriore 20-30 minuti dopo aver aggiunto l&#39;ultima parte del solfato di ammonio per garantire la completa equilibrio. DPS rimarranno solubile, mentre le proteine ​​contaminanti saranno precipitare dalla soluzione. </li><li> Rimuovere proteine ​​precipitate mediante centrifugazione a 20.000 xg per 30 min a 4 ° C e salvare il supernatante. </li><li> Lentamente aggiungere un supplemento di 227 mg di solfato di ammonio per ml di surnatante di raggiungere il 94% di saturazione e mescolare per 20-30 minuti per garantire la completa equilibrio. DPS precipitati a questa concentrazione di sale e possono essere raccolti per centrifugazione a 20.000 xg per 30 min a 4 ° C. DPS saranno nel pellet, il supernatante può essere scartato. Il pellet può essere conservato a -80 ° C se desIred. </li><li> Risospendere il Dps contenenti pellet in Resuspension Buffer (50 mM HEPES-KOH, pH 7.5, 150 mM NaCl, 0,1 mM EDTA). Regolare il volume del campione di 2,5 ml. </li><li> Scambio di tamponi per allontanare il solfato di ammonio, utilizzando una colonna di filtrazione PD-10 gel. Equilibrare il letto con gel 25 ml Resuspension Buffer. Applicare il ml 2,5 di Dps campione alla parte superiore della colonna PD-10 e lasciare in ammollo dentro Eliminare il flusso continuo. Raccogliere Dps eluendo con 3,5 ml Resuspension Buffer. </li><li> Centrifugare il buffer a scambio campione a 16.000 xg per 10 min a 4 ° C per rimuovere i componenti insolubili, e diluire con 6-10 ml di tampone di diluizione (50 mM HEPES-KOH, pH 7,5, 0,1 mM EDTA) per garantire che la concentrazione di sale è abbastanza basso per il Dps di legare alla colonna SP Sepharose. Campioni particolarmente concentrate di DPS può diventare meno solubili su di diluizione in un tampone inferiore-sale, come evidenziato da nebulosità del liquido, ma possono essere completamente resolubilized con l&#39;aggiunta di un piccolo Amount 5 M NaCl (un ulteriore 10-20 mm). </li><li> Equilibrare un ml SP Sepharose colonna flusso veloce con 30 SP tampone A (50 mM HEPES-KOH, pH 7.5, 50 mM NaCl, 0,1 mM EDTA). Esegui tampone attraverso la colonna a 1,5-2 ml / min, con un limite di pressione di 0,3 MPa su sfondo. Caricare il campione attraverso la colonna. Lavare con SP tampone A fino a quando le OD 280 traccia ritorna alla linea di base, e scartare il flow-through. Eseguire un gradiente lineare da 150 ml da 0 al 100% Tampone B, mentre la raccolta 2 frazioni ml. DPS eluiscano in un forte picco intorno al 50% B, sebbene variazione del 10% è possibile. </li><li> Eseguire le frazioni di eluizione su un gel SDS-PAGE 15%, e verificare la presenza di contaminazione da DNA misurando il rapporto di 280 OD 260 / OD utilizzando uno spettrofotometro. Il gel deve mostrare solo una banda di circa 19 kDa, e la OD 260 / OD 280 è in genere intorno a 0.7. La piscina per i più puri Dps frazioni. Utilizzare un gruppo filtro centrifugo (come il Amicon Ultra Filtration Unit) con un10K peso molecolare di cut-off per concentrare il DPS e scambiarli in un buffer di memoria (50 mM HEPES-KOH, pH 7.5, 50 mM di NaCl). Aliquota della Dps purificati, e congelare in azoto liquido per la conservazione a -80 ° C. </li></ol> 2. Protezione DNA Assay Il saggio coinvolge più passaggi sensibili al tempo e dovrebbe essere cronometrato al secondo per ottenere risultati riproducibili. Molti ingredienti e le fasi di pipettaggio sono coinvolti, in modo da un tavolo di pipettaggio (vedi tabella 1) Si consiglia di tenere traccia dei passaggi e dei volumi. Il tempo totale richiesto per il dosaggio è non più di 3 ore. <ol><li> Misurare 30 ml di acqua in un flacone ermetico; lavare l&#39;acqua con N 2 per 10 min (usando due aghi siringa, una nel fluido, uno sopra di esso) per rimuovere l&#39;ossigeno dalla soluzione. Aggiungi 0,0168 g FeSO4 • 7H 2 O per l&#39;acqua per ottenere una soluzione madre di 2 mm. Si chiude la fiala e lavare con azoto per 5 minuti di più, poi mescolare. La soluzione deve esserechiaro, se ogni accenno di giallo è rilevabile, preparare una nuova soluzione di ferro. </li><li> Fare un 100 mm H 2 O la soluzione 2 (10,87 ml di 9.2MH 2 O 2 in 1 ml di acqua); tenere in ghiaccio e al riparo dalla luce avvolgendolo in un foglio di alluminio. Utilizzare solo per circa 1-2 ore dopo la preparazione a causa di decadimento spontaneo. Il perossido di idrogeno è disponibile similmente soggetta a guasti, che possono essere parzialmente alleviate corretta conservazione (al buio a 4 ° C). Sono consigliati controlli regolari della concentrazione stock di misurare i 240 OD. </li><li> Scongelare una aliquota di purificata Dps rapidamente in un bagno d&#39;acqua a temperatura ambiente, e tenere in ghiaccio. Centrifugare per 10 secondi a 4000 xg in un tavolo picocentrifuge ad aggregati precipitato. Misurare la concentrazione dopo centrifugazione utilizzando uno spettrofotometro (OD 280 = 1,547 indica una concentrazione di 100 mM Div. monomero). </li><li> Diluire il DNA lineare da alta concentrazione di magazzino utilizzando 12x tampone di reazione (1M MOPS-KOH pH 7,0, NaCl 1 M) ad una concentrazione di 100 ng / mL. DNA lineare è usata per rendere più facile quantificazione, ma il DNA plasmidico può essere utilizzato come bene. </li><li> Pipettare 1 ml di miscela di DNA-buffer in un tubo di PCR. Aggiungere abbastanza acqua nella reazione in modo che il volume di reazione finale (meno SDS) sarà di 12 microlitri. Tale importo deve essere calcolato in anticipo utilizzando la tabella di pipettaggio. Aggiungi Dps ad una concentrazione finale dodecamero di 3 micron. Incubare per 15 min a temperatura ambiente per permettere Div. di legarsi al DNA. </li><li> Aggiungere abbastanza 2 mM soluzione FeSO 4 per raggiungere la concentrazione desiderata. Un tipico esperimento utilizza un intervallo di concentrazioni da 0 a 1 mM FeSO4. Concentrazioni maggiori causerà più ampio degradazione del DNA, ma può anche portare alla formazione di precipitati ferrico nella miscela di reazione. Aggiungere rapidamente 1 mM microlitri della soluzione di H 2 O 2 100 (a concentrazione finale 10 mM), e incubare a temperatura ambiente per 5 minuti per consentire la FentonMediata reazione di degradazione di prendere posto. </li><li> Aggiungere 0,8 ml di 20% SDS (sodio dodecil solfato). Mescolare ed incubare a 85 ° C per 5 minuti per distruggere i complessi Dps-DNA. La SDS destabilizza il complesso Dps-DNA e impedisce spostamenti indesiderati gel per il DNA, che migliora la facilità di quantificazione. Un passo opzionale è fermare la reazione catalitica degradando il perossido di idrogeno in prodotti non tossici. Per le condizioni descritte in questo protocollo, questa fase non è necessaria per ottenere una vasta gamma di degradazione del DNA. </li><li> Incubare in ghiaccio per 1 min, aggiungere colorante di caricamento, e caricare su un gel di agarosio. Esegui il gel, e colorare per DNA usando bromuro di etidio. Il gel deve essere colorato post-elettroforesi anziché prima dell&#39;elettroforesi, per evitare che la SDS di interferire con la distribuzione bromuro di etidio nel gel. </li></ol>

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Il processo di purificazione Div. come descritto in questo articolo è molto robusto. La purezza è stata assai alta (&gt; 99%); altre proteine ​​appaiono su SDS-PAGE gel come bande visibili. Nonostante questo, alcuni lotti di Dps purificati sembrano avere attività nucleasi, come dimostra la degradazione del DNA parziale quando incubate con concentrazioni molto elevate di Dps. Ciò potrebbe indicare la presenza di DNasi altamente attivi a bassa concentrazione che non siamo riusciti a rimuovere attraverso la purific...

Organismi aerobici devono lottare costantemente con le specie reattive dell&#39;ossigeno che possono danneggiare il DNA e altre macromolecole biologiche cruciali. Uno strumento potente per contrastare gli effetti tossici del danno ossidativo è la proteina legante il DNA dalle cellule affamate (DPS). Fin dalla sua scoperta nel 1992 da fame E. coli cultura 1, Div. è stata identificata in più di 300 specie di batteri e archeobatteri 2. Upregulation massiccia di Dps durante la fase stazionaria lo rende il più altamente espresso proteina nucleoide-associato di E. coli in condizioni di fame 3, 4. Inoltre, Dps ha mostrato di preservare sia la vitalità batterica e l&#39;integrità del DNA durante molte diverse sollecitazioni, tra cui la fame, ad alta concentrazione di ferro, l&#39;esposizione alla luce UV, shock termico, e lo stress ossidativo 5, 6. Strutturalmente, Dps auto-soci in un complesso stabile omo-oligomeri di 12 monomeri, which assemblare in un guscio vuoto sferico. Il ~ 4.5 nm-wide cavità interna è accessibile al solvente esterno attraverso pori che permettono il passaggio di piccole molecole 7, e può sequestrare metalli come ferro mineralizzati 8. L&#39;effetto protettivo del Dps deriva dalle sue molteplici attività biochimiche, che comprendono non specifico DNA binding 1, l&#39;attività ferrossidasica e deposito di ferro 8. Studio dettagliato delle benefiche attività biochimiche del Dps richiede prima la sua purificazione. DPS purificazione è una procedura complessa, come PS devono essere separati non solo da altre proteine, ma anche da qualsiasi DNA legato 7. Nostro processo di purificazione ottimizzato utilizza molte tecniche comuni, costituito da due colonne di scambio ionico e di un passo precipitazione di solfato di ammonio. Sono necessari molti scambi di buffer, come Dps alta concentrazione possono precipitare dalla soluzione in condizioni di basso contenuto di sale. Una volta Dps proteina è stata purificata, Esso può essere applicato a saggi che misurano direttamente la sua attività ferrossidasica 8, stechiometria DNA-binding 9, e meccanismi di ferro vincolante 10. Dps purificate ha anche altre applicazioni potenziali. La struttura sferica cava stabile di DPS è stato utilizzato come impalcatura per la memorizzazione di particelle idrofobe all&#39;interno della cavità proteina 11 e anche come camera di reazione per sintetizzare nuove nanoparticelle magnetiche 12. La capacità protettiva del Dps di mediare i danni derivanti da specie reattive dell&#39;ossigeno può essere chiaramente e direttamente dimostrato utilizzando il saggio di protezione del DNA 13, 14. In questa procedura in vitro, specie radicali sono prodotti quando ferro catalizza H 2 O 2 degradazione attraverso la chimica Fenton. Questi radicali danneggiare direttamente DNA presente nella reazione e possono degradare completamente a concentrazioni elevate. Due principali attività DPS può sia contrastare direttamente gli effetti di Fentproduzione di radicali on-mediata. Dps abbassa la concentrazione di ferro catalitico mediante mineralizzazione, consumando il perossido di idrogeno disponibile nel processo. Inoltre, Dps legame al DNA possono potenzialmente proteggerlo fisicamente dai danni dei radicali e lo condensa in un volume più piccolo con superficie meno reattivo. La combinazione di queste due proprietà rende il saggio di protezione DNA adatto per lo scopo di misurare l&#39;attività protettiva Div.. Il saggio di protezione Il DNA è molto versatile e può essere utilizzato per una varietà di applicazioni oltre caratterizzazione Div.. I danni dei radicali è una comune forma di stress nelle cellule, e molte proteine ​​e sostanze chimiche diverse sono utilizzati per contrastarla. Il principio generale del saggio, l&#39;integrità del DNA utilizzando come marker per danni radicale, può essere utilizzato in combinazione con qualsiasi reazione producono radicali o un agente di contrasto. Tra gli altri, il saggio è stato usato con successo per determinare le proprietà anti-ossidantidi K. Estratto paniculata per l&#39;industria alimentare 15, per caratterizzare gli effetti di acido urico a danno idrossile mediazione 16, e di acquisire nuove informazioni sulla funzione delle proteine ​​Fur regolatore trascrizionale 17. Nonostante i numerosi usi del saggio in articoli pubblicati, abbiamo scoperto che molti di ottimizzazione e risoluzione dei problemi sono stati richiesti, il che rende l&#39;impostazione del test per la prima volta un processo inutilmente faticoso per molti ricercatori. Il protocollo che presentiamo in questo articolo si propone di rimuovere questa barriera per l&#39;ingresso.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 </tr>
 <tr>
 <td>BL21(DE3)pLysS competent cells</td>
 <td>Promega</td>
 <td>L1195</td>
 </tr>
 <tr>
 <td>pET-17b DNA</td>
 <td>EMD-Millipore</td>
 <td>69663-3</td>
 </tr>
 <tr>
 <td>LB broth powder</td>
 <td>Sigma-Aldrich</td>
 <td>L3022</td>
 </tr>
 <tr>
 <td>Ampicillin sodium salt</td>
 <td>Sigma-Aldrich</td>
 <td>A0166</td>
 </tr>
 <tr>
 <td>Chloramphenicol</td>
 <td>Sigma-Aldrich</td>
 <td>C0378</td>
 </tr>
 <tr>
 <td>IPTG</td>
 <td>Sigma-Aldrich</td>
 <td>I6758</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>BDH</td>
 <td>441476L</td>
 </tr>
 <tr>
 <td>Potassium hydroxide</td>
 <td>Merck</td>
 <td>105033</td>
 </tr>
 <tr>
 <td>Sodium chloride</td>
 <td>VWR</td>
 <td>443824T</td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Sigma-Aldrich</td>
 <td>E9884</td>
 </tr>
 <tr>
 <td>Protease Inhibitor Cocktail Set III</td>
 <td>EMD-Millipore</td>
 <td>539134</td>
 </tr>
 <tr>
 <td>DEAE-Sepharose</td>
 <td>Sigma-Aldrich</td>
 <td>DFF100</td>
 </tr>
 <tr>
 <td>Ammonium sulphate</td>
 <td>Sigma-Aldrich</td>
 <td>A4418</td>
 </tr>
 <tr>
 <td>PD-10 Desalting Columns</td>
 <td>GE Healthcare LS</td>
 <td>17-0851-01</td>
 </tr>
 <tr>
 <td>SP-Sepharose</td>
 <td>Sigma-Aldrich</td>
 <td>S1799</td>
 </tr>
 <tr>
 <td>Amicon Ultra Centr. Filter (10K MWCO)</td>
 <td>Millipore</td>
 <td>UFC901024</td>
 </tr>
 <tr>
 <td>Ferrous Sulphate heptahydrate</td>
 <td>Sigma-Aldrich</td>
 <td>F8048</td>
 </tr>
 <tr>
 <td>Hydrogen peroxide solution</td>
 <td>Sigma-Aldrich</td>
 <td>216763</td>
 </tr>
 <tr>
 <td>MOPS</td>
 <td>Calbiochem</td>
 <td>475898</td>
 </tr>
 <tr>
 <td>SDS solution</td>
 <td>Bio-Rad</td>
 <td>161-0418</td>
 </tr>
 <tr>
 <td>Ethidium bromide</td>
 <td>Sigma-Aldrich</td>
 <td>E1510</td>
 </tr>
 <tr>
 <td>Equipment</td>
 <td>Company</td>
 <td>Model</td>
 </tr>
 <tr>
 <td>Static incubator</td>
 <td>Hettich</td>
 <td>INE500</td>
 </tr>
 <tr>
 <td>Shaking Incubator </td>
 <td>New Brunswick Sc.</td>
 <td>Inova 44</td>
 </tr>
 <tr>
 <td>Cooled centrifuge</td>
 <td>Beckman Coulter</td>
 <td>Avanti J-E</td>
 </tr>
 <tr>
 <td>Table-top centrifuge</td>
 <td>Eppendorf</td>
 <td>5424</td>
 </tr>
 <tr>
 <td>Cell disrupter</td>
 <td>Constant Systems Ltd.</td>
 <td>TS2/40/AA/AA</td>
 </tr>
 <tr>
 <td>FPLC Purifier</td>
 <td>General Electric</td>
 <td>AKTA</td>
 </tr>
 <tr>
 <td>Airtight vials</td>
 <td>Cole-Parmer</td>
 <td>EW-08918-85</td>
 </tr>
 <tr>
 <td>Syringe needles</td>
 <td>BD</td>
 <td>305128</td>
 </tr>
 <tr>
 <td>Pipettes</td>
 <td>Eppendorf</td>
 <td>Z683779-1EA, Z683795-1EA</td>
 </tr>
 </tbody>
</table>

application of an in vitro dna protection assay to visualize stress mediation properties of the dps protein

Lo stress ossidativo è un sottoprodotto inevitabile della vita aerobica. L&#39;ossigeno molecolare è essenziale per il metabolismo terrestre, ma partecipa anche a molte reazioni dannose all&#39;interno di organismi viventi. La combinazione di metabolismo aerobico e di ferro, che è un altro composto essenziale per la vita, è sufficiente a produrre radicali attraverso la chimica di Fenton e degradare componenti cellulari. Degradazione del DNA è probabilmente il processo più dannosa che coinvolgono radicali intracellulari, come la riparazione del DNA è tutt&#39;altro che banale. Il saggio presentato in questo articolo offre una tecnica quantitativa per misurare e visualizzare l&#39;effetto delle molecole ed enzimi sui danni al DNA radicale-mediata. Il saggio di protezione del DNA è un semplice, rapido, e robusto strumento per la caratterizzazione in vitro delle proprietà protettive delle proteine ​​o prodotti chimici. Esso consiste nell&#39;esporre DNA ad una reazione ossidativa dannosa e aggiungendo concentrazioni varianti del composto di interesse. La riduzione o l&#39;aumento di danno al DNA in funzione della concentrazione del composto è quindi visualizzato mediante elettroforesi su gel. In questo articolo si dimostra la tecnica del saggio di protezione del DNA misurando le proprietà protettive della proteina legante il DNA dalle cellule affamate (DPS). DPS è un mini-ferritina che è utilizzato da più di 300 specie di batteri per combattere con forza di stress ambientale. Qui vi presentiamo il protocollo di purificazione Dps e le condizioni di analisi ottimizzate per valutare la protezione del DNA dal Dps.

Il processo di purificazione per il Dps qui descritto è molto riproducibile. Dps purificazione secondo il protocollo descritto, utilizzando 2 L di E. cultura coli come punto di partenza, sarà tipicamente resa 2,5 ml di proteina contenente Div. 12 a concentrazioni comprese tra 5 e 12 mM. Tempi di induzione più lunghi (4 ore) sembrano ridurre questa variabilità. Purezza proteina è costantemente al di sopra del 99%, come evidenziato da SDS-PAGE gel (Figura 1). Il livello d...

Watch this Scientific Journal Video about Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein at JoVE.com

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

La proteina di legame al DNA dalle cellule affamate (DPS) gioca un ruolo cruciale nella lotta contro lo stress batterica. Questo articolo discute la purificazione<em&gt; E. coli</em&gt; PS e il protocollo per un<em&gt; In vitro</em&gt; Test dimostrando protezione Dps-mediata di DNA dalla degradazione dalle specie reattive dell&#39;ossigeno.

Applicazione di un<em&gt; In vitro</em&gt; DNA Protection Assay per visualizzare stress Mediazione Proprietà del Dps Protein

Oxidative stress is an unavoidable byproduct of  aerobic life.  Molecular oxygen is  essential for terrestrial metabolism, but it also takes part in many ...

application-an-vitro-dna-protection-assay-to-visualize-stress

Research

JoVE Journal

Biology

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

11.1K Views.  Delft University of Technology. La proteina di legame al DNA dalle cellule affamate (DPS) gioca un ruolo cruciale nella lotta contro lo stress batterica. Questo articolo discute la purificazione E. coli PS e il protocollo per un In vitro Test dimostrando protezione Dps-mediata di DNA dalla degradazione dalle specie reattive dell'ossigeno.

Video: Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

Un In vitro FluoroBlok Tumore Assay Invasion

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

Ricerca

Biologia

Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein

Transient kinetic analysis is indispensable for understanding the workings of biological macromolecules, since this approach yields mechanistic information including active site concentrations and intrinsic rate constants that govern macromolecular function. In case of enzymes, for example, transient or pre-steady state measurements identify and characterize individual events in the reaction pathway, whereas steady state measurements only yield overall catalytic efficiency and specificity. Individual events such as protein-protein or protein-ligand interactions and rate-limiting conformational changes often occur in the millisecond timescale, and can be measured directly by stopped-flow and chemical-quench flow methods. Given an optical signal such as fluorescence, stopped-flow serves as a powerful and accessible tool for monitoring reaction progress from substrate binding to product release and catalytic turnover1,2.
Here, we report application of stopped-flow kinetics to probe the mechanism of action of Msh2-Msh6, a eukaryotic DNA repair protein that recognizes base-pair mismatches and insertion/deletion loops in DNA and signals mismatch repair (MMR)3-5. In doing so, Msh2-Msh6 increases the accuracy of DNA replication by three orders of magnitude (error frequency decreases from ~10-6 to10-9 bases), and thus helps preserve genomic integrity. Not surprisingly, defective human Msh2-Msh6 function is associated with hereditary non-polyposis colon cancer and other sporadic cancers6-8. In order to understand the mechanism of action of this critical DNA metabolic protein, we are probing the dynamics of Msh2-Msh6 interaction with mismatched DNA as well as the ATPase activity that fuels its actions in MMR. DNA binding is measured by rapidly mixing Msh2-Msh6 with DNA containing a 2-aminopurine (2-Ap) fluorophore adjacent to a G:T mismatch and monitoring the resulting increase in 2-aminopurine fluorescence in real time. DNA dissociation is measured by mixing pre-formed Msh2-Msh6 G:T(2-Ap) mismatch complex with unlabeled trap DNA and monitoring decrease in fluorescence over time9. Pre-steady state ATPase kinetics are measured by the change in fluorescence of 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin)-labeled Phosphate Binding Protein (MDCC-PBP) on binding phosphate (Pi) released by Msh2-Msh6 following ATP hydrolysis9,10.
The data reveal rapid binding of Msh2-Msh6 to a G:T mismatch and formation of a long-lived Msh2-Msh6 G:T complex, which in turn results in suppression of ATP hydrolysis and stabilization of the protein in an ATP-bound form. The reaction kinetics provide clear support for the hypothesis that ATP-bound Msh2-Msh6 signals DNA repair on binding a mismatched base pair in the double helix.
F. Noah Biro and Jie Zhai contributed to this paper equally.

Msh2-Msh6 is responsible for initiating repair of replication errors in DNA. Here we present a transient kinetics approach towards understanding how this critical protein works. The report illustrates stopped-flow experiments for measuring the coupled DNA binding and ATPase kinetics underlying Msh2-Msh6 mechanism of action in DNA repair.

Applicazione di metodi Stopped-flow Cinetica per Indagare sulla Meccanismo d&#39;azione di una proteina di riparazione del DNA

Transient kinetic analysis is indispensable for understanding the workings of biological macromolecules, since this approach yields mechanistic ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

Saggi biofisici per sondare le proprietà meccaniche del nucleo della cellula Interphase: Strain supporto Applicazione e manipolazione microago

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

A lungo termine del test di tossicità letale con il crostaceo Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

In vitro metilazione Assay per studiare proteine ​​Arginina Metilazione

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively inert because it has two unpaired electrons located in different pi * anti-bonding orbitals. These two electrons have parallel spins, meaning they rotate in the same direction about their own axes. This is why the oxygen molecule is not very reactive. Activation of oxygen may occur by two different mechanisms; either through reduction via one electron at a time (monovalent reduction), or through the absorption of sufficient energy to reverse the spin of one of the unpaired electrons. This results in the production of reactive oxidative species (ROS). There are a number of ways in which the human body eliminates ROS in its physiological state. If ROS production exceeds the repair capacity, oxidative stress results and damages different molecules. There are many different methods by which oxidative stress can be measured. This manuscript focuses on one of the methods named cell gel electrophoresis, also known as &#8220;comet assay&#8221; which allows measurement of DNA breaks. If all factors known to cause DNA damage, other than oxidative stress are kept constant, the amount of DNA damage measured by comet assay is a good parameter of oxidative stress. The principle is simple and relies upon the fact that DNA molecules are negatively charged. An intact DNA molecule has such a large size that it does not migrate during electrophoresis. DNA breaks, however, if present result in smaller fragments which move in the electrical field towards the anode. Smaller fragments migrate faster. As the fragments have different sizes the final result of the electrophoresis is not a distinct line but rather a continuum with the shape of a comet. The system allows a quantification of the resulting &#8220;comet&#8221; and thus of the DNA breaks in the cell.

Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.

Comet Assay come una misura indiretta della sistemica Stress ossidativo

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of &#947;- and &#946;-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called &#947;H2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled &#947;H2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors.

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented.

Misurazione danno al DNA e ripristina in mouse Splenociti Dopo cronica<em&gt; In Vivo</em&gt; L&#39;esposizione a dosi molto basse di beta e gamma Radiazioni

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Immunoprecipitazione della cromatina Saggio per la identificazione di Arabidopsis interazioni proteina-DNA<em&gt; In Vivo</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

L&#39;Assemblea e l&#39;applicazione di &quot;Shear Anelli &#39;: A Novel endoteliale modello per Orbital, unidirezionale e periodica del fluido di flusso e lo stress di taglio

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. To improve the mating efficiency of B. Xylophilus and to increase the chances of mating observation, virgin adults were cultured and used for the investigation. Eggs were obtained by keeping the nematodes in water and allowing the females to lay eggs for 10 min. The second-stage juveniles (J2) were synchronized by incubating the eggs for 24 h at 25 &#176;C in the dark, and the early J4 were obtained by culturing the J2 with grey mold, Botrytis cinerea, for another 52 h. At this time point, most J4 nematodes could be clearly distinguished as being male or female using their genital morphology. The male and female J4 were collected and cultured separately in two different Petri dishes for 24 h to get virgin adult nematodes. A virgin male and a virgin female were paired in a drop of water in the well of a concave slide. The mating behavior was filmed with a video recorder under a stereomicroscope. The whole period of the mating process was 82.8 &#177;3.91 min (mean &#177;SE) and could be divided into 4 different phases: searching, contacting, copulating, and lingering. The mean minutes of duration were 21.8 &#177; 2.0, 28.0 &#177; 1.9, 25.8 &#177; 0.7 and 7.2 &#177; 0.5, respectively. Eleven sub-behaviors were described: cruising, approaching, encountering, touching, hooping, locating, attaching, ejaculating, separating, quiescence, and roaming. Interestingly, obvious intra-sexual competition was observed when one female was grouped with 3 males or one male with 3 females. This protocol is useful and valuable, not only in investigating the mating behavior of B. xylophilus, but also in acting as a reference for ethological studies of other nematodes.

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A protocol for investigating the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, is presented. Behavioral features of B. xylophilus are described in the mating process.

Osservazione e quantificazione di comportamento di accoppiamento nel nematode del pino,<em&gt; Bursaphelenchus xylophilus</em

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. ...