Sviluppo del NU-ELISA metodo è stato sostenuto da NIH concedere RO1AG023687.

1. Colture cellulari di mammifero NU-ELISA può essere eseguita su qualsiasi tipo di cellula di mammifero che si può crescere in coltura. Noi preferiamo preparare da moderati a grandi lotti di cellule in modo che nucleosomi possono essere isolati in quantità sufficiente per preparare diversi piatti identico caricato ELISA, consentendo test con diversi anticorpi (Abs). Le scale cultura seguenti forniscono ampio materiale: Per il mouse cellule staminali embyronic, crescere uno o due 15 tavole cm di cellule senza cellule alimentatore. Per i fibroblasti, crescere 5-10 15 piatti cm a confluenza. 2. Isolamento dei nuclei Nota: Tutti i passi sono sul ghiaccio con pre-raffreddata buffer, ad eccezione di quanto indicato. <ol><li> Trypsinize celle con 3 tripsina ml per piastra, combinare e aggiungere 20 ml di ghiacciata PBS / butirrato. Centrifugare a 1000 rpm per 5 min. </li><li> Risospendere le cellule in 10 ml di PBS / butirrato e centrifugare a 1000 rpm per 5 min. </li><li> Risospendere in 4 ml di tampone di lisi con inibitori della proteasi (Sigma-Aldrich P-8340). Dounce omogeneizzare 20 colpi di pestello di tipo B sul ghiaccio. </li><li> Centrifugare a 2000g per 10 minuti a 4 ° C. (Il supernatante contiene citoplasma, che non è necessario per questo protocollo, ma può essere salvato per altri usi se lo si desidera). </li><li> Risospendere il pellet in 2 ml di ghiaccio freddo lavare tampone C (con inibitori della proteasi). </li><li> Strato di materiale risospeso in 5 ml cuscino di saccarosio 30%, poi centrifugare a 2400g per 5 minuti in un rotore oscillante. I nuclei migreranno attraverso cuscino, e detriti rimane a livello di interfaccia. </li><li> Rimuovere tutti i volumi di liquido con attenzione e risospendere i nuclei in 250 microlitri di tampone di lavaggio ghiacciata C + inibitori della proteasi. </li></ol> 3. Isolamento dei nucleosomi da parte in situ Micrococcal nucleasi (MNase) digestione Noi utilizziamo una procedura in cui viene digerito cromatina in situ all&#39;interno di nuclei da infondendo loro MNase, seguito da un trattamento ipotonico a guidare senza nucleosomi intatti nel sopranatante. <ol><li> Aggiungere 3 ml 0,1 M CaCl 2 al campione. Mettere in 37 blocchi ° C Calore. Permettono di assumere 37 ° C di temperatura. </li><li> Aggiungere 2 unità MNase (Micrococcal nucleasi, Sigma-Aldrich, sciolto in 2 units/10μl nel buffer MNase), poi incubare a 37 ° C per 12 minuti, con frequenti rimontaggi con una punta di pipetta. </li><li> Aggiungere 6 ml di 0,5 M Na-EDTA, pH 8,0 per arrestare la reazione. Mettere in ghiaccio. </li><li> Centrifugare a 2000g per 4 minuti, scartare il surnatante. Risospendere il pellet in 300 ml 0,2 mm Na-EDTA. Conservare in ghiaccio per 1 ora con occasionali pipeting gentile. (Queste condizioni ipotoniche liberare nucleosomi privi di nuclei). </li><li> Centrifugare a 3000 g per 4 minuti a 4 ° C. Salvare il surnatante, che contiene nucleosomi libero, sul ghiaccio. </li><li> Risospendere il pellet di nuovo con 300 ml 0,2 mm Na-EDTA. Conservare in ghiaccio per 1 ora con occasionali pipeting gentile. </li><li> Centrifugare a 3000g per 4 min a 4 ° C. Conservare il surnatante e si combinano con il sopranatante primo passo 5. I preparativi mononucleosome risultante può essere aliquotati e conservati a -80 ° C. </li></ol> Nota: la quantità di cromatina può essere crudamente quantificato misurando l&#39;assorbanza a 260 nm di un campione di 10 microlitri aggiunto a 990 ml di acqua. A 260 = 10 (dopo aggiustamento per i 1 / 100 di diluizione) corrisponde a circa 1mg/ml della cromatina. Inoltre, durante la procedura di cui sopra, piccoli campioni possono essere conservati e successivamente analizzate per il loro contenuto di DNA per monitorare la qualità e l&#39;estensione della digestioni MNase, che dovrebbe prevalentemente contengono DNA di 146 bp di lunghezza. Laddering è indicativo di digestione MNase incompleta. Nota: Fig.1 contiene una sintesi diagramma di isolamento nucleare e digestioni MNase passi. 4. Nucleosoma-ELISA (NU-ELISA) Rileviamo PTM sulle frazioni contenenti nativo nuclesosomes intatto che sono stati immobilizzati su 96 e piastre microtiter ELISA. Per ogni campione, facciamo una serie di 2-diluizioni, e questi sono rivestiti su pozzi in triplice copia. <ol><li> Cappotto piastre Maxisorp notte a 4 ° C con 50 microlitri / pozzetto di nucleosomi diluito in tampone di rivestimento. Suggerito diluizioni duplice serie di nucleosomi sono preparati dalla prima riga per riga in basso con l&#39;aggiunta di 0,1 mg, 0,05 mg, 0.025 mg, 0,0125 mg, mcg 0,00625, 0,00313 mg, 0,00156 mg, con tampone di rivestimento solo (0 cromatina mcg) nella parte inferiore fila. (Nota: comprende piastra sono necessari per questo passaggio.) </li><li> Al mattino, lavare i piatti 4 volte con 200 l / pozzetto PBS/0.5% di Tween-20 a temperatura ambiente (RT) per un totale di 10 min con una rondella di piatto. </li><li> Blocco 1 ora a temperatura ambiente con 100 l / pozzetto PBS/0.05% di Tween-20 / 5% BSA. </li><li> Rimuovere il tampone di bloccaggio agitando la piastra capovolta vivacementepiù di un lavandino. Coprire e conservare le piastre a -20 ° C, o andare direttamente alla fase successiva. </li><li> Aggiungere 1 ° Abs in un volume di 50 microlitri / ben diluito 1:1000 (o secondo necessità) in PBS/0.05% Tween-20 / 5% BSA, incubare a RT per 1 ora su un rotatore. </li><li> Lavare le piastre 4 volte con 200 l / pozzetto PBS/0.5% di Tween-20 a temperatura ambiente e per un totale di 10 min in una lavatrice piatto. </li><li> Aggiungi rafano perossidasi di cavallo (HRP)-coniugato 2 ° Abs, diluito 1:5000 (o secondo necessità) in PBS/0.05% Tween-20 / 5% BSA a temperatura ambiente per un&#39;ora su un rotatore. </li><li> Lavare le piastre 4 volte con 200 l / pozzetto PBS/0.5% di Tween-20. Ogni lavaggio è a temperatura ambiente e per un totale di 10 minuti con una rondella di piatto. </li><li> Sviluppare i piatti aggiungendo 50 l di substrato TMB ad ogni pozzetto per 10 minuti a temperatura ambiente. Bloccare la reazione aggiungendo 50 microlitri / pozzetto di 2N H 2 SO 4 e leggere le piastre 450 nm. (Suggerimento: Centrifugare a 1500 rpm per 2 minuti utilizzando un rotore piatto per dissipare eventuali bolle d&#39;aria nei pozzetti prima assorbanze lettura). </li><li> Esportare le letture ad un foglio elettronico per analisi quantitative e statistiche. </li></ol> Reagenti <table border="1"><tbody><tr><td> PBS / butirrato </td></tr><tr><td> 135 mM NaCl </td></tr><tr><td> 2,5 mM KCl </td></tr><tr><td> 8 mM Na 2 HPO 4 </td></tr><tr><td> 1,5 mM KH 2 PO 4 </td></tr><tr><td> 10 mM Na-butirrato </td></tr></tbody></table><table border="1"><tbody><tr><td> Lysis Buffer </td></tr><tr><td> 250 mm di saccarosio </td></tr><tr><td> 10 mM Tris-HCl, pH 7.4 </td></tr><tr><td> 10 mM Na-butirrato </td></tr><tr><td> 4 mM MgCl 2 </td></tr><tr><td> 0,1% Triton X-100 </td></tr></tbody></table><table border="1"><tbody><tr><td> Tampone di lavaggio C </td></tr><tr><td> 250 mm di saccarosio </td></tr><tr><td> 10 mM Tris-HCl, pH 7.4 </td></tr><tr><td> 10 mM Na-butirrato </td></tr><tr><td> 4 mM MgCl 2 </td></tr></tbody></table><table border="1"><tbody><tr><td> Saccarosio Cuscino </td></tr><tr><td> 30% (w / v) di saccarosio nel tampone di lavaggio C </td></tr></tbody></table><table border="1"><tbody><tr><td> Microccocal nucleasi Buffer </td></tr><tr><td> 5 NaPO mm 4 tampone, pH 7,0 </td></tr><tr><td> 0,025 mm CaCl 2 </td></tr></tbody></table><table border="1"><tbody><tr><td> Rivestimento tampone </td></tr><tr><td> Soluzione A + 80ml 170ml Soluzione B + 250ml dH 2 O </td></tr><tr><td> Soluzione A: 0,2 M Na 2 CO 3 </td></tr><tr><td> Soluzione B: 0,2 M NaHCO 3 </td></tr></tbody></table> 5. Rappresentante Risultati Utilizzando il NU-ELISA metodo, una serie di diversi piatti identici vengono preparati che possono essere caricati con cromatina preparato nella forma di mononucleosomes. Noi di solito preparare serie di identico-caricati piastre in modo che ciascuno può essere sondato con diversi anticorpi specifici anti-PTM (Abs). Abbiamo anche sempre preparare un piatto che può essere sondato con un Ab che rileva istoni senza riguardo al loro controllo totale stato di caricamento della cromatina modifica. Questo controllo è essenziale per poi confrontare quantitativamente contenuto PTM dei nucleosomi preparati da campioni diversi. Per quantificare i livelli di PTM all&#39;interno di un campione di cromatina dato che correggere i valori di assorbanza prima con questo approccio: in primo luogo, si sottrae ogni segnale di fondo utilizzando i valori ottenuti da pozzetti di controllo che non contengono cromatina (sfondo di solito è trascurabile). Abbiamo poi standardizzare PTM-specifici segnali di NU-ELISA risultati ottenuti da una piastra identico carico che è stato testato con un Ab che rileva nucleosomi indipendentemente dal loro stato di modifica. (Abbiamo usato Abs policlonali specifici per gli istoni H2A e H2B per questo scopo). Siamo quindi determinare medie e le varianze per ogni diluizione della cromatina, e utilizzare i dati ottenuti dalla parte lineare del test ELISA. Esempi dettagliati di NU-ELISA analisi matematiche e statistiche sono state precedentemente riportate 4 <img src="/files/ftp_upload/2593/2593fig1.jpg" alt="Figura 1"/> Figura 1. Schematica diagam di NU-ELISA passi. A. Le cellule di mammifero vengono raccolte; B. I nuclei sono separati dalle cellule da Dounce homogenization, C. Turbativa cellule vengono caricati sulla cima di un 30% w / v cuscino di saccarosio, D. Dopo la centrifugazione, i nuclei si depositano nel pellet, E, F. cromatina sono digeriti in situ principalmente per mononucleomes da MNase, G, H , io. mononucleosomes solubili sono estratte da un trattamento ipotonico e centrifugazione ripetuta di residui di materiale nucleare. Mononucleosomes J. da diversi campioni (con etichetta samp. 1 e 2 in questo caso) sono rivestiti in pozzetti in serie di identico caricati piatti e interrogati con PTM-specifici Abs e PTM-Abs indipendente per determinare il carico totale della cromatina. Rappresentazione K. di livelli di segnale di rilevazione differenziale in due campioni che si differenziano per il loro contenuto PTM, ancora simile contenuto complessivo della cromatina, come giudicato da anti-H2B immunoreattività.

<ol>
	<li>Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F., Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+nucleosome+core+particle+at+2.8+A+resolution.">Crystal structure of the nucleosome core particle at 2.8 A resolution.</a> Nature. 389, 251-260 (1997).</li><li>Jenuwein, T., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+the+histone+code.">Translating the histone code.</a> Science. 293, 1074-1080 (2001).</li><li>Turner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+acetylation+and+an+epigenetic+code.">Histone acetylation and an epigenetic code.</a> Bioessays. 22, 836-845 (2000).</li><li>Dai, B., Rasmussen, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+epiproteomic+signatures+distinguish+embryonic+stem+cells+from+differentiated+cells.">Global epiproteomic signatures distinguish embryonic stem cells from differentiated cells.</a> Stem Cells. 25, 2567-2574 (2007).</li><li>Tanasijevic, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+accumulation+of+epigen+etic+heterogeneity+during+human+ES+cell+culture.">Progressive accumulation of epigen etic heterogeneity during human ES cell culture.</a> Epigenetics. 4, 330-338 (2009).</li><li>Thomas, J. O., Gould, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+fractionation+of+chromatin+and+linker+histones.">Isolation and fractionation of chromatin and linker histones.</a> Chromatin: a practical approach. , 12-14 (1998).</li><li>Thorne, A. W., Cary, P. D., Crane-Robinson, C., Gould, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+separation+of+core+histones+and+non-histone+chromosomal+proteins.">Extraction and separation of core histones and non-histone chromosomal proteins.</a> Chromatin: a practical approach. , 38-39 (1998).</li></ol>

Nessun conflitto di interessi dichiarati.

NU-ELISA fornisce un metodo per accertare lo stato globale di PTM nucleosoma presenti all&#39;interno di un particolare tipo di cellula. NU-ELISA studi hanno dimostrato che gli stati globali modifica nucleosoma differiscono in confronto dei tipi cellulari divergenti 4. Inoltre, NU-ELISA profili PTM cambiare quando le cellule sono esposte ad agenti modulatori epigenetici come tricostatina-A o quando il mouse cellule staminali embyronic sono differenziati 4. Il metodo è stato applicato con successo allo studio della cromatina delle cellule staminali embrionali umane 5. E &#39;importante notare la NU-ELISA, nella sua forma attuale, in grado di rilevare solo la firma composito di PTM presenti all&#39;interno della somma totale dei epigenoma cellulare. Questo si differenzia notevolmente da genome-wide immunoprecipitazione della cromatina, che può determinare la distribuzione di un PTM specifico attraverso specifici loci genetici. Le fasi iniziali del NU-ELISA procedure sono adattate da precedenti metodi per isolare mononucleosomes da nuclei mammiferi 6,7. Questi metodi adattato fornire un espediente per ottenere mononucleosomes intatto di alta qualità dalle cellule dei mammiferi, e le frazioni risultanti sono complete nel loro contenuto cromatina, ma contengono una grande quantità di ulteriore materiale nucleare. Dato che sono utilizzati Abs, contaminando non nucleosomal materiale è ben tollerato nel NU-test ELISA, a differenza di massa-spec approcci che richiedono una maggiore purezza. Tuttavia, NU-ELISA è più sensibile e quantitativo di western blotting 4, fornendo così buone alternative ai western e spettrometro di massa per la rilevazione dei PTM nucleosomal.

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti </th></tr><tr><td> Micrococcal Nucleae </td><td> Sigma </td><td> N3755 </td><td> Fai aliquote di 2 tampone U/10μl, conservati a -20 ° C </td></tr><tr><td> Nunc Maxisorp microtitolazione Piastre </td><td> Pescatore </td><td> 12-565-135 </td><td> Copre targhetta adesiva possono essere ordinati anche tramite Fisher </td></tr><tr><td> Piastra Rondella automatica </td><td></td><td></td><td></td></tr><tr><td> 1-Step TMB-substrato ELISA </td><td> Perforare </td><td> 34028 </td><td> Conservare a 4 ° C pre-caldo per RT prima dell&#39;uso o come indicato sulla scheda tecnica </td></tr><tr><td> Lettore di micropiastre </td><td> Bio-Rad lettore di micropiastre (modello: 680) </td><td></td><td> La maggior parte dei modelli colorimetrici lettore di piastre sono adatti </td></tr></tbody></table>

detection of post-translational modifications on native intact nucleosomes by elisa

Il genoma degli eucarioti esiste come cromatina che contiene sia il DNA e le proteine. L&#39;unità fondamentale della cromatina è il nucleosoma, che contiene 146 paia di basi di DNA associate con due ciascuno degli istoni H2A, H2B, H3 e H4 1. N-terminale code degli istoni sono ricche di lisina e arginina e vengono modificati post-trascrizionalmente da acetilazione, metilazione e altre modificazioni post-traduzionali (PTM). La configurazione del PTM nucleosomi può influenzare l&#39;attività trascrizionale del DNA associate, fornendo così un modo di regolazione genica che è epigenetici in natura 2,3. Abbiamo sviluppato un metodo chiamato nucleosoma ELISA (NU-ELISA) per determinare quantitativamente firme PTM globale dei nucleosomi estratto dalle cellule. NU-ELISA è più sensibile e quantitativo di western blotting, ed è utile per interrogare lo stato epiproteomic di tipi cellulari specifici. Questo articolo di giornale video mostra le procedure dettagliate per eseguire NU-ELISA analisi.

Watch this Scientific Journal Video about Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA at JoVE.com

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

Nucleosoma ELISA (NU-ELISA) è un metodo sensibile e quantitativa per rilevare modelli globali di modificazioni post-traduzionali nelle preparazioni dei nativi, nucleosomi intatto. Queste modifiche includono methylations, acetylations e fosforilazioni a specifici residui di aminoacidi degli istoni, e quindi NU-ELISA fornisce un test globale proteomica della complessivo stati modificazione della cromatina di tipi cellulari specifici.

Rilevazione di modificazioni post-traduzionali in nativo nucleosomi intatto da ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

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Research

JoVE Journal

Biology

17.2K Views.  Stanford University. Nucleosoma ELISA (NU-ELISA) è un metodo sensibile e quantitativa per rilevare modelli globali di modificazioni post-traduzionali nelle preparazioni dei nativi, nucleosomi intatto. Queste modifiche includono methylations, acetylations e fosforilazioni a specifici residui di aminoacidi degli istoni, e quindi NU-ELISA fornisce un test globale proteomica della complessivo stati modificazione della cromatina di tipi cellulari specifici.

Video: Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

Detection of Functional Matrix Metalloproteinases by Zymography

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11.
Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.

This protocol describes an activity-based assay for detecting matrix metalloproteinases in culture supernatants or body fluids.

Rilevamento delle metalloproteinasi della matrice funzionale di zimografia

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been ...

Ricerca

Biologia

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Analizzando la funzione di GTPasi Small Microiniezione di plasmidi in cellule epiteliali polarizzate

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Detection of Histone Modifications in Plant Leaves

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7. 
Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4 photosynthesis in maize5,11 and systemic immunity in Arabidopsis3.

A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.

Rilevamento di modifiche Histone in foglie delle piante

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Germogliamento lievito proteine ​​estrazione e purificazione per lo Studio della funzione, interazioni e modificazioni post-traduzionali

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Identificazione delle modificazioni post-traduzionali di proteine ​​vegetali Complessi

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

L&#39;isolamento di regioni genomiche specifici e l&#39;identificazione di molecole da Associated enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

En Face Detection di ossido nitrico e superossido in strato endoteliale delle arterie Intact

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

High-throughput CRISPR vettore Costruzione e caratterizzazione di modifiche del DNA da Generazione di Roots pomodoro Peloso

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

Flusso di lavoro completo per l&#39;analisi dell&#39;istone modificazioni post-traduzionali Utilizzando Bottom-up Spettrometria di Massa: Da istone estrazione all&#39;analisi dei dati

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 &#176;C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy.

Three heat precipitation methods are presented that effectively remove more than 90% of host cell proteins (HCPs) from tobacco extracts prior to any other purification step. The plant HCPs irreversibly aggregate at temperatures above 60 &#176;C.

Confronto di tabacco metodi Host cellulare della proteina di rimozione da Blanching Piante intatte o mediante trattamento termico di estratti

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...