Don Pettine

<html> 1. Deglycosylation enzimatica <ol><li> Utilizzare provette PCR per ridurre al minimo la perdita d&#39;acqua a causa dell&#39;evaporazione. Etichettare una serie di tubi 1 a 7. </li><li> Scongelare il buffer 10X G7, il buffer glicoproteina 10X denaturazione e il 10% NP-40 e picchiettare delicatamente i tubi per mescolare il contenuto. Conservare a temperatura ambiente. </li><li> Posizionare l&#39;enzima contenente fiale sul ghiaccio. Cercare di minimizzare disgelo / cicli di congelamento. </li><li> Sciogliere il contenuto della fiala hCGβ (150 mg) in 600 ml di dH 2 O e tenere sul ghiaccio. </li><li> Preparare 1 ml di tampone 1X G7 diluendo lo stock 10X in dH 2 O. </li><li> Diluire 0,5 ml di PNGase F in 25 microlitri di buffer 1X G7 e mantenere sul ghiaccio. </li><li> Preparare l&#39;impasto exoglycosidase (mix EG) combinando due microlitri ognuna delle α-N-Acetylgalactosaminidase, α1-2 Fucosidase, β1-3 galattosidasi, e α1-3, 6 galattosidasi. </li><li> Istituito provette PCR come indicato: </li></ol>ak &quot;&gt; <table border="1"><tbody><tr><td> Tubo / campione # </td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 7 </td></tr><tr><td> hCGβ (0.25mg/ml) </td><td> 9μl </td><td> 9μl </td><td> 9μl </td><td> 9μl </td><td> - </td><td> - </td><td> - </td></tr><tr><td> Glicoproteina 10X denaturazione Buffer </td><td> 1ml </td><td> 1ml </td><td> 1ml </td><td> 1ml </td><td> 1ml </td><td> 1ml </td><td> 1ml </td></tr><tr><td> dH 2 O </td><td> - </td><td> - </td><td> - </td><td> - </td><td> 9μl </td><td> 9μl </td><td> 9μl </td></tr></tbody></table><ol start="9"><li> Cap i tubi, mescolare delicatamente e mettere intermociclatore, chiudere il coperchio e denaturare le proteine ​​incubando 10 minuti a 94 ° C, seguito da un 4 ° premuto C (utilizzando tubi di PCR in un termociclatore impedisce notevolmente l&#39;evaporazione in campioni di piccolo volume). </li><li> Rimuovere i tubi dal termociclatore e centrifugare per rimuovere l&#39;eventuale condensa visibile. </li><li> Aggiungere i seguenti reagenti come indicato: </li></ol><table border="1"><tbody><tr><td> Esempio # </td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 7 </td></tr><tr><td> 10% NP-40 </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td></tr><tr><td> 10X Buffer G7 </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5 &amp; mu; L </td><td> 2.5μl </td></tr><tr><td> PNGase F 1:50 dil. </td><td> - </td><td> 2μl </td><td> - </td><td> - </td><td> 2μl </td><td> - </td><td> - </td></tr><tr><td> Deglycosylation Mix </td><td> - </td><td> - </td><td> 2μl </td><td> 2μl </td><td> - </td><td> 2μl </td><td> 2μl </td></tr><tr><td> Mix EG </td><td> - </td><td> - </td><td> - </td><td> 2μl </td><td> - </td><td> - </td><td> 2μl </td></tr><tr><td> dH 2 O </td><td> 10μl </td><td> 8μl </td><td> 8μl </td><td> 6μl </td><td> 8μl </td><td> 8μl </td><td> 6μl </td></tr><tr><td> Reazione vol totale. </td><td> 25 μ l </td><td> 25 μ l </td><td> 25 μ l </td><td> 25 μ l &lt;/ Td&gt; <td> 25 μ l </td><td> 25 μ l </td><td> 25 μ l </td></tr></tbody></table><ol start="12"><li> Chiudere i tubi PCR utilizzando tappi nuovi (scartare quelli usati dal momento che non si inseriscono correttamente dopo un ciclo di incubazione). </li><li> Mescolare i tubi picchiettando delicatamente 4 volte e poi girare i contenuti verso il basso. </li><li> Mettere le provette nel termociclatore e incubare a 37 ° C per 4 ore poi il raffreddamento dei campioni a 4 ° C. </li></ol> 2. SDS-PAGE di campioni deglicosilata <ol><li> Preparare 130 microlitri fresca 3X riducendo tampone di caricamento SDS con l&#39;aggiunta di 4 ml di DTT 1.25m. </li><li> Aggiungere 12,5 ml di preparato 3X riducendo il tampone di caricamento SDS per ogni campione. </li><li> Chiudere i tubi con tappi nuovi e picchiettare delicatamente i tubi per mescolare. </li><li> Incubare le provette in un termociclatore a 94 ° C per 5 minuti e poi raffreddare a 4 ° C. </li><li> Carico 30 ml di ogni campione e 10 ml di marker di proteine ​​su un 10-20% Tris-Glicina gel. Salva il resto del campione per la parte 3.1. Carico 10μl di Protein Marker ColorPlus Prestained. </li><li> Elettroforesi il gel a 130 volt a temperatura ambiente fino a quando il fronte del colorante è vicino alla parte inferiore del gel. </li><li> Quando il gel ha terminato l&#39;esecuzione, rimuovere il gel dal cast e metterlo in una piccola scatola di plastica con sufficiente Coomasie macchia blu per coprire il gel. </li><li> Macchiare il gel per 1 ora e scuotendo con cautela. </li><li> Lavare il gel tre volte per 30 minuti in 50 ml di soluzione Decolorare. </li><li> Registrare le immagini utilizzando un transilluminatore a luce bianca o scanner. In alternativa, il gel può essere asciugati tra fogli di cellophane in una cornice. </li></ol> 3. Pro-Q Smeralda 300 per il rilevamento delle proteine ​​glicosilate in SDS-PAGE gel <ol><li> In parallelo con il gel in 2.1), caricare il resto dei campioni su un 10-20% Tris-Glicina gel. Carico 10 e mu; L di proteine ​​marker ColorPlus Prestained. </li><li> Mentre il gel è in esecuzione sciogliere il Pro-Q reagente Smeraldo con DMF e preparare la correzione magazzino, lavaggio e ossidanti soluzioni per la Pro-Q Emerald 300 macchia seguendo il manuale del prodotto fornito con il kit. </li><li> Quando l&#39;elettroforesi, rimuovere il gel da cast e metterlo in una scatola di plastica. </li><li> Fissare il gel con l&#39;aggiunta di 100 ml di soluzione Fix e lasciandola notte a temperatura ambiente e scuotendo con cautela. </li><li> Lavare il gel con 100 ml di soluzione di lavaggio per 10 a 20 minuti a temperatura ambiente con agitazione gentile. Ripetere il lavaggio con soluzione di lavaggio fresca. </li><li> Ossidare i carboidrati tramite l&#39;incubazione del gel con leggera agitazione per 30 minuti in 25 ml di soluzione ossidante. </li><li> Lavare il gel come descritto al punto 3.5. </li><li> Mentre il gel è fresco lavaggio preparare Pro-Q Emerald 300 macchia aggiungendo 500 microlitri della Pro-Q Emerald 300 disciolti in soluzione reagente punto 3.2 a 25 ml dicolorazione buffer fornito nel kit. </li><li> Macchiare il gel con l&#39;aggiunta di 25 ml della macchia preparato al passo 3.8 e incubazione al buio scuotendo con cautela per 90 a 120 minuti. </li><li> Ripetere le due fasi di lavaggio descritto al punto 3.5. </li><li> Registra le immagini con un transilluminatore UV a 300nm. Utilizzare il marcatore 80 kDa, che viene etichettato con Pro-Q verde smeraldo, di sovrapporre l&#39;immagine ai raggi UV per una fotografia luce bianca del gel che mostra la scala prestained. </li><li> Confrontare le immagini del gel colorato Coomasie al passo 2,10 con la Pro-Q gel colorato Smeraldo a partire dal punto 3.11. </li></ol> 4. Rappresentante risultati I cambiamenti nella migrazione proteina enzimatica dopo deglycosylation sono illustrati nella Figura 2. Confrontare il campione di controllo (pannello A, corsia 1) con il trattamento F PNGase (rimozione di N-glicani, corsia 2), e Mix Deglycosylation (PNGase F; più endo-e exoglycosidases per rimuovere l&#39;O-glicani, corsia 3). Nessun ulteriore riduzionedelle dimensioni è visto dopo la digestione con glicosidasi aggiuntivi (corsia 4). Oltre ad un cambiamento di massa, bande diventano più nitide come glicani vengono rimossi. Una banda di esecuzione con l&#39;indicatore 17 kDa (asterisco) rappresenta completamente deglicosilata hCGβ polipeptidiche (MW: 16 kDa). Altre band potrebbero derivare da deglycosylation incompleti o dalle proteine ​​più non identificati presenti nel campione hCGβ (vedi corsia 1). Corsie 5 a 7 (controlli) mostrano la banda della glicosidasi. La colorazione glicoproteina dal verde smeraldo è mostrato nel pannello B. Questo reagente ossida e macchie di tutti i glicani presenti in una molecola proteica. Quindi l&#39;intensità del segnale diminuisce hCGβ è enzimaticamente deglicosilata (corsia 1 a corsia 4). Il segnale residuo a corsie 3 e 4 indicano la presenza di motivi glicani, che sono resistenti agli enzimi utilizzati. Le glicosidasi aggiuntivi utilizzati in corsia 4 rimuovere residui di qualche zucchero in più: la migrazione delle proteine ​​è lo stesso, mauna leggera riduzione di intensità della colorazione può essere vista. Frazioni di zucchero resistenti non erano presenti in tutte le specie delle proteine: alcune band non sono stati rilevati dal verde smeraldo (assente nell&#39;immagine UV, tra parentesi), indicando che sono stati ampiamente deglicosilata. Ulteriori dati supportano la conclusione che hCGβ è eterogeneo glicosilata. La banda inferiore sulla corsia 2 (freccia) è debole l&#39;immagine verde smeraldo, mentre la banda superiore di corsia 2 è luminoso, che indica che molti gruppi glicani sono ancora presenti. Questi dati supportano la conclusione che hCGβ ricombinante espresso in cellule di topo contiene glicoforme più 9. Queste glicoforme diverse sono dovute alla eterogeneità intrinseca della glicosilazione dove alcuni polipeptidi non ricevono una glicani in ogni luogo del consenso e / o alcuni glicani sono estesi, mentre altri anche sulla stessa proteina non lo sono. <img alt="Figura 1" src="/files/ftp_upload/3749/3749fig1.jpg" /> Figura 1.Modelli di glicosilazione tipico per secreta o superficie cellulare glicoproteine. <img alt="Figura 2" src="/files/ftp_upload/3749/3749fig2.jpg" /> Figura 2. SDS-PAGE gel che mostra deglycosylation enzimatica di hCGβ. Pannello A mostra una colorazione blu, mentre Coomasie Panel B mostra i risultati di Pro-Q Smeralda 300 per la visualizzazione delle proteine ​​glicosilate. Numero del campione: 1, il controllo hCGβ, 2, PNGase F digestione, 3, Mix digestione Deglycosylation, 4, Mix Deglycosylation più digestione exoglycosidases, campioni 5 a 7 sono i controlli reattivi (O-Glyc, O-glicosidasi, GNA, β-N -acetilglucosaminidasi; Fucosidase Gal / Fuc, β1-3 galattosidasi, α1-3, 6 galattosidasi, e α1-2; GalNA, α-N-Acetylglalactosaminidase, Neu, neuraminidasi; PNGase, F PNGase)

<ol>
	<li>Spiro, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+glycosylation:+nature,+distribution,+enzymatic+formation,+and+disease+implications+of+glycopeptide+bonds.">Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds.</a> Glycobiology. 12 (4), 43-56 (2002).</li><li>Ohtsubo, K., Marth, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosylation+in+Cellular+Mechanisms+of+Health+and+Disease.">Glycosylation in Cellular Mechanisms of Health and Disease.</a> Cell. 126, 855-867 (2006).</li><li>Arnold, J. N., Wormald, M. R., Sim, R. B., Rudd, P. M., Dwek, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+glycosylation+on+the+biological+function+and+structure+of+human+immunoglobulins.">The impact of glycosylation on the biological function and structure of human immunoglobulins.</a> Annual Review of Immunology. 25, 21-50 (2007).</li><li>Mitra, N., Sinha, S., Ramya, T. N. C., Surolia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-linked+oligosaccharides+as+outfitters+for+glycoprotein+folding,+form+and+function.">N-linked oligosaccharides as outfitters for glycoprotein folding, form and function.</a> Trends in Biochemical Sciences. 31, 156-163 (2006).</li><li>Carlsen, R. B., Bahl, O. P., Swaminathan, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+chorionic+gonadotropin.+Linear+amino+acid+sequence+of+the+beta+subunit.">Human chorionic gonadotropin. Linear amino acid sequence of the beta subunit.</a> Journal of Biological Chemistry. 248 (19), 6810-6812 (1973).</li><li>Tarentino, A. L., Plummer, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deglycosylation+of+Asparagine-linked+Glycans+by+PNGaseF.">Deglycosylation of Asparagine-linked Glycans by PNGaseF.</a> Trends in Glycoscience and Glycotechnology. 5 (23), 163-170 (1993).</li><li>Tretter, V., Altmann, F., Marz, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide-N4-(N-acetyl-&#946;-glucosaminyl)asparagine+amidase+F+cannot+release+glycans+with+fucose+attached+&#945;1-3+to+the+asparagine-linked+N-acetylglucosamine+residue.">Peptide-N4-(N-acetyl-&#946;-glucosaminyl)asparagine amidase F cannot release glycans with fucose attached &#945;1-3 to the asparagine-linked N-acetylglucosamine residue.</a> European Journal of Biochemistry. 199, 647-652 (1991).</li><li>Koutsioulis, D., Landry, D., Guthrie, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+endo-&#945;-N-acetylgalactosaminidases+with+broader+substrate+specificity.">Novel endo-&#945;-N-acetylgalactosaminidases with broader substrate specificity.</a> Glycobiology. 18 (10), 799-805 (2008).</li><li>Thakur, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+the+glycoforms+of+the+intact+alpha+subunit+of+recombinant+human+chorionic+gonadotropin+by+high-resolution+capillary+electrophoresis-mass+spectrometry.">Profiling the glycoforms of the intact alpha subunit of recombinant human chorionic gonadotropin by high-resolution capillary electrophoresis-mass spectrometry.</a> Analytical Chemistry. 81 (2), 8900-8907 (2009).</li><li>Park, S., Lee, M. R., Shin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbohydrate+microarrays+as+powerful+tools+in+studies+of+carbohydrate-mediated+biological+processes.">Carbohydrate microarrays as powerful tools in studies of carbohydrate-mediated biological processes.</a> Chemical Communications. 37, 4389-4399 (2008).</li><li>Zachara, N. E., Vosseller, K., Hart, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+analysis+of+proteins+modified+by+O-linked+N-acetylglucosamine.">Detection and analysis of proteins modified by O-linked N-acetylglucosamine.</a> Current Protocols in Molecular Biology. 95, 17-17 (2011).</li><li>Varki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Essentials of Glycobiology. , (2008).</li></ol>

Gli autori sono impiegati da New England Biolabs, che produce molti dei reagenti utilizzati in questo articolo.

Il metodo descritto qui utilizzando deglycosylation enzimatica e SDS-PAGE in grado di fornire preziose informazioni sullo stato glicosilazione di una proteina di interesse, mentre glycan reagenti specifici per agevolare l&#39;interpretazione dei dati. Questo protocollo è stato progettato per gli studi iniziali di glicosilazione delle proteine ​​ed è particolarmente adatto per secretoria e glicoproteine ​​di membrana di cellule di mammifero: gli enzimi scelto in questo caso specifico rimuovere tutti gli N-glicani, e o N-glicani plus e gli zuccheri più comuni estendere e costituiscono il nucleo di O-glicani. Glicosidasi hanno il vantaggio aggiuntivo di essere mite, rispetto ai metodi chimici deglycosylation, preservare l&#39;integrità di zuccheri e di spina dorsale delle proteine. Per chiarire il tasso di occupazione (che gli aminoacidi sono glicosilata), l&#39;estensione della glicosilazione, o per determinare la struttura fine di glicani, tecniche più sofisticate come la mspettrometria di culo, la cromatografia liquida o NMR sono obbligatori. A causa della sua semplicità, diversi passi in questo protocollo può essere regolata, sostituiti e / o combinati per soddisfare varie esigenze sperimentali. Tuttavia, al fine di ottenere risultati che possono essere chiaramente interpretato, è importante capire i punti di forza e limiti. In primo luogo, la specificità e la purezza delle glicosidasi sono cruciali: solo gli enzimi ben caratterizzati testati per evitare di proteasi e altre attività inquinanti dovrebbero essere usati. Purtroppo, non c&#39;è uno standard per la definizione di unità di enzimi glicosidasi, l&#39;utilizzatore dovrebbe determinare la sostituzione appropriata in base alle specifiche del costruttore. In secondo luogo, una scelta accurata di rilevazione è necessario: a) i reagenti colorazione delle proteine ​​sono utili solo se i risultati deglycosylation in un cambiamento significativo nella massa molecolare. Tale risultato chiaro come mostrato qui non è sempre ottenuta. In altri casi, abbiamo visto mi anormaleintegrazione dopo deglycosylation (lontano dal peso molecolare previsto, o la migrazione ancora più lento). Questo fenomeno non è ben compreso, ma si può dire che ogni cambiamento nella migrazione è la prova che la proteina è stata deglicosilata. B) individuazione di zucchero con anticorpi rappresenta una sfida unica limitando la loro applicabilità. E &#39;stato molto difficile per generare generale anticorpi anti-glicani, che solitamente sono sollevate contro un obiettivo complesso glicani, che limita il loro uso. Inoltre, diverse monoclonali anti-glicani anticorpi schermo indesiderato reattività incrociata 10. C) Le lectine (proteine ​​con zucchero affinità intrinseca) sono adatti per il rilevamento di zucchero, più danno visione sulla struttura glicani. Tuttavia, non tutti hanno una specificità stretto, e molti sono solo parzialmente caratterizzato (nel senso che avrebbero potuto affinità sconosciuto). Come un segnale positivo colorazione lectina conseguenza, non fornisce indicazioni, la prova, la presenza di un given zucchero. d) kit di etichettatura chimica (sulla base di periodato ossidazione degli zuccheri) sono il metodo di scelta per colorare tutti gli glicoproteine, e quindi ben si adatta a seguire deglycosylation. Quando si elabora un campione sconosciuto, è una buona pratica includere controlli glicoproteina. Fetuina è un N prontamente disponibili - e O-glicoproteina, gonadotropina corionica (entrambe le subunità) è anche una buona scelta. Sieroalbumina bovina (BSA) può essere utilizzato come controllo negativo. Tuttavia, va notato che alcuni non glicosilata proteine ​​reagiscono un po &#39;con la Pro-Q Smeralda 300, in particolare quando utilizzati ad alte concentrazioni. Non glicosilata standard di peso molecolare, come il marcatore proteina prestained utilizzati in questo protocollo, hanno il vantaggio di visualizzare le bande taglienti. Solo per caso è una di queste proteine ​​(80 kDa) reattiva a Pro-Q Smeralda 300. Pertanto, l&#39;utente potrebbe desiderare di eseguire una scala standard glicoproteina invece, come la Candy-Cane da Invitrogen. Infine, semplice-gel rilevazione delle glicoproteine ​​nucleocytosolic (che sono modificati con un singolo O-GlcNAc) è possibile con l&#39;utilizzo di un anticorpo monoclonale, insieme β-N-acetilglucosaminidasi per il controllo della specificità 11. La descrizione di questa tecnica è al di là del scopo di questo articolo, ma dovrebbe essere menzionato come glicosidasi sono strumenti utili per lo studio di molte altre glicoproteine ​​e glicoconiugati presenti nelle cellule. Un trattamento completo di tutte le forme conosciute di glicosilazione può essere trovato nella seconda edizione di &quot;Fondamenti di Glycobiology&quot;, disponibile gratuitamente on-line al scaffale NCBI (ID scaffale: NBK1908; PMID: 20301239) 12.

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Β gonadotropina corionica umana </td><td> Sigma Aldrich </td><td> C6572 </td><td></td></tr><tr><td> PCR Tubes </td><td> VWR </td><td> 20170-004 </td><td></td></tr><tr><td> PCR Termociclatore </td><td> Applied Biosystems </td><td> 4359659 </td><td></td></tr><tr><td> PNGase F </td><td> New England Biolabs </td><td> P0704 </td><td> Fornito con tamponi </td></tr><tr><td> Proteine ​​Deglycosylation Mix </td><td> New England Biolabs </td><td> P6039 </td><td> Fornito con tamponi </td></tr><tr><td> α-N-Acetylglalactosaminidase </td><td> New England Biolabs </td><td> P0734 </td><td> Fornito con tampone </td></tr><tr><td> α1-2 Fucosidase </td><td> New England Biolabs </td><td> P0724 </td><td> Fornito con tampone </td></tr><tr><td> α1-3, 6 galattosidasi </td><td> New England Biolabs </td><td> P0731 </td><td> Fornito con tampone </td></tr><tr><td> β1-3 galattosidasi </td><td> New England Biolabs </td><td> P0726 </td><td> Fornito con tampone </td></tr><tr><td> Buffer 10X G7 (500 mM fosfato di sodio) </td><td> New England Biolabs </td><td> --- </td><td> Fornito con PNGaseF o degl Mix </td></tr><tr><td> Glicoproteina Buffer 10X denaturazione (5% SDS, 400 mM DTT) </td><td> New England Biolabs </td><td> --- </td><td> Fornito con o degl PNGaseF Mix </td></tr><tr><td> 10% NP-40 </td><td> New England Biolabs </td><td> --- </td><td> Fornito con PNGaseF o degl Mix </td></tr><tr><td> 3X SDS tampone di caricamento (187,5 mM Tris-HCl pH 6.8, 6% SDS, 30% glicerolo, 0,03% Bromophenol blu) </td><td> New England Biolabs </td><td> B7703 </td><td> Fornito con 1,25 m DTT </td></tr><tr><td> ColorPlus Prestained Proteine ​​Marker </td><td> New England Biolabs </td><td> P7709 </td><td></td></tr><tr><td> 10-20% Tris-Glicina Multigel </td><td> Cosmo Bio Co. </td><td> DCB-414893 </td><td></td></tr><tr><td> Cassette Elettroforesi Unità </td><td> Cosmo Bio Co. </td><td> DCB-303111 </td><td></td></tr><tr><td> Elettroforesi di alimentazione EPS di alimentazione 301 </td><td> GE Healthcare </td><td> 18-1130-01 </td><td></td></tr><tr><td> Pro-Q Emerald 300 Kit Glicoproteina Stain </td><td> Invitrogen </td><td> P-21857 </td><td></td></tr><tr><td> Brilliant Blue R </td><td> Sigma Aldrich </td><td> B0149 </td><td></td></tr><tr><td> AlphaImager HP System </td><td> Cellula Biosciences </td><td> 92-13823-00 </td><td></td></tr></tbody></table>

identification and characterization of protein glycosylation using specific endo- and exoglycosidases

Glicosilazione, l&#39;aggiunta di zuccheri legati covalentemente, è un importante modificazione post-traslazionale di proteine ​​che possono influenzare significativamente i processi, come l&#39;adesione delle cellule, il traffico molecolare, di liquidazione, e la trasduzione del segnale 1-4. Negli eucarioti, le modifiche glicosilazione più comuni nella via secretoria sono aggiunte a residui di asparagina consenso (N-linked), oppure a residui di serina o treonina (O-linked) (Figura 1). Avvio di N-glicano sintesi è altamente conservato negli eucarioti, mentre il prodotto finale può variare notevolmente tra specie diverse, tessuti, o proteine. Alcuni glicani non vengono modificati (&quot;alto mannosio N-glicani&quot;) o vengono ulteriormente elaborati nel Golgi (&quot;complesso di N-glicani&quot;). Diversità maggiore è trovato per O-glicani, che inizia con un comune N-acetilgalattosamina (GalNAc) residui nelle cellule animali, ma si differenziano per organismi inferiori 1. ENT &quot;&gt; L &#39;analisi dettagliata della glicosilazione delle proteine ​​è un settore a sé e richiede ampie risorse e competenze per eseguire correttamente. tuttavia una serie di enzimi disponibili che gli zuccheri rimuovere (glicosidasi) rende possibile avere una idea generale dello stato di glicosilazione . una proteina in un ambiente di laboratorio standard Qui illustrare l&#39;uso di glicosidasi per l&#39;analisi di una glicoproteina modello. ricombinante gonadotropina corionica umana beta (hCGβ), che trasporta due N-glicani e quattro O-glicani 5 La tecnica richiede solo semplice strumentazione e materiale di consumo tipico, e può essere facilmente adattato alle analisi dei campioni glicoproteina multipli. Numerosi enzimi possono essere utilizzati in parallelo per studiare una glicoproteina. PNGase F è in grado di rimuovere quasi tutti i tipi di N-glicani legate 6,7. Per O-glicani, non c&#39;è enzima disponibile che può fendere un oligosaccaride intatto dal secoloe proteine ​​spina dorsale. Invece, O-glicani sono rifilati da exoglycosidases a breve un nucleo, che viene facilmente rimosso da O-glicosidasi. Il Mix Deglycosylation Protein contiene PNGase F, O-glicosidasi, neuraminidasi (sialidasi), β1-4 galattosidasi e β-N-acetilglucosaminidasi. E &#39;utilizzato per rimuovere simultaneamente N-glicani e qualche O-glicani 8. Infine, il Mix Deglycosylation è stata integrata con una miscela di exoglycosidases altri (α-N-Acetylgalactosaminidase, α1-2 Fucosidase, α1, 3, 6 galattosidasi, e β1-3 galattosidasi), che aiutano a rimuovere monosaccaridi altrimenti resistenti che potrebbero essere presenti in alcune O-glicani. SDS-PAGE/Coomasie blu è usato per visualizzare le differenze in migrazione proteine ​​prima e dopo il trattamento glicosidasi. In aggiunta, uno zucchero metodo specifico colorazione, ProQ Smeraldo-300, mostra come il segnale diminuisce glicani sono successively rimosso. Questo protocollo è progettato per l&#39;analisi di piccole quantità di glicoproteina (da 0,5 a 2 mg), anche se deglycosylation enzimatica può essere scalata fino a ospitare grandi quantità di proteine, se necessario.

Watch this Scientific Journal Video about Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases at JoVE.com

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Utilizzando glicosidasi specifico per rimuovere gli zuccheri da glicoproteine ​​seguita da SDS-PAGE è un metodo valido per individuare modifiche glycan su campioni di proteine ​​ed è una buona scelta per gli studi Glycobiology iniziale. Seguenti modifiche deglycosylation possono essere rilevati come spostamenti della mobilità gel o con la colorazione glycan reagenti sensibili.

Identificazione e caratterizzazione di glicosilazione delle proteine ​​con specifiche endo-e exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

identification-characterization-protein-glycosylation-using-specific

Research

JoVE Journal

Biology

36.4K Views.  New England Biolabs. Utilizzando glicosidasi specifico per rimuovere gli zuccheri da glicoproteine ​​seguita da SDS-PAGE è un metodo valido per individuare modifiche glycan su campioni di proteine ​​ed è una buona scelta per gli studi Glycobiology iniziale. Seguenti modifiche deglycosylation possono essere rilevati come spostamenti della mobilità gel o con la colorazione glycan reagenti sensibili.

Video: Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

L&#39;identificazione di complessi proteici con la proteomica quantitativa in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Ricerca

Biologia

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Utilizzando il sistema GELFREE 8100 Frazionamento per peso molecolare basata su Frazionamento con recupero della fase liquida

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

Individuazione di partner proteine ​​interagenti mediante Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

Anticorpo Microarray chimicamente bloccato per Multiplexed high-throughput Profiling di glicosilazione delle proteine ​​specifico in campioni complessi

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

Metodo per l&#39;isolamento e l&#39;identificazione di mRNA, microRNA e componenti proteiche dei complessi Ribonucleoproteina da estratti cellulari con RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Identificazione di complessi proteici in<em&gt; Escherichia coli</em&gt; Con sequenziale purificazione per affinità Peptide in combinazione con Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

Generazione, purificazione e caratterizzazione di specie DISC1 Cell-invasive Protein

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.

Pyrosequencing per identificazione microbica e caratterizzazione

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate ...

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 ...

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

We describe a modified method for membrane fraction isolation from human cells and sample preparation for the detection of SCAP N-glycosylation and total protein by using western blot. We further introduce a GFP-labeling method to monitor SCAP trafficking using confocal microscopy. This protocol can be used in regular biology laboratories.

Identificazione e caratterizzazione di glicosilazione delle proteine ​​con specifiche endo-e exoglycosidases

Identificazione e caratterizzazione di glicosilazione delle proteine con specifiche endo-e exoglycosidases