The authors would like to acknowledge Dr. John Sheehan and Dr. Lubna Abdullah for their guidance and mentoring that were central in the completion of this work. This work was supported by funds from the National Institutes of Health (P01HL108808, UH2HL123645) and the Cystic Fibrosis Foundation Therapeutics, Inc. (EHRE07XX0). Kathryn Ramsey is supported by an NHMRC Early Career Research fellowship.

1. Preparare buffer per Mucin Gel Western Blotting <ol><li> Preparare 1 litro di 50x TAE (Tris-acetato-EDTA) buffer. <ol><li> In 700 ml di acqua distillata (dH 2 O) aggiungere 242 g di Tris base (0,4 M), 57,1 g di acido acetico glaciale (pesare liquido) (0,2 M) e 14.61 g di acido etilendiamminotetraacetico (EDTA) (50 mM). </li><li> Regolare il pH a 8,0 e rendere il volume fino a 1 litro con dH 2 O. </li></ol></li><li> Preparare 10 ml di 10x tampone di caricamento. <ol><li> Preparare 5 ml di tampone 1x TAE. A tale scopo, aggiungere 5 ml di glicerolo (50%), 25 mg di blu di bromofenolo (0,25%) e 100 mg di sodio dodecil solfato (SDS) (1%). </li></ol></li><li> Preparate 2 litri di tampone 20x di sodio citrato di soluzione salina (SSC). <ol><li> In 1,5 litri di dH 2 O, aggiungere 350,6 g di NaCl (3 M) e 176,4 g di citrato trisodico (Na 3 C 6 H 5 O 7) (0,3 M). Regolare il pH a 7,0 e portare il volume a 2 litri con dH 2 O. </li></ol></li><li> Preparare 1 litro di 1x tampone TAE-0.1% SDS. <ol><li> Aggiungere 20 ml di 50x TAE a 980 ml ​​di dH 2 O. Aggiungere 1 g di SDS per ottenere una soluzione 0,1% SDS-TAE. </li></ol></li></ol> 2. TAE-SDS agarosio gel Preparazione <ol><li> (. Vale a dire, 150 ml) Versare un volume sufficiente di 1x TAE-0,1% tampone SDS in un pallone di Erlenmeyer nel microonde per fare un 5 - gel di 7 millimetri di spessore. Aggiungere 0,8% (1,2 g) di polvere di agarosio. </li><li> Forno a microonde pallone in intervalli di 30 sec con vorticoso intermittente fino agarosio è completamente sciolto (in genere 1 - 3 min). Usare tamponi mano calda durante questo processo. Fare attenzione di ebollizione eruttiva mentre vorticoso. </li><li> Lasciare che la soluzione di agarosio raffreddare per 5 - 10 minuti. Nota: La soluzione di agarosio sarà pronto per essere versato quando fondo del pallone può essere lasciato sul palmo per 5 sec. </li><li> Preparare vassoio colata elettroforesi (10 x 15 cm) sigillando i bordi con nastro e sistemazione ben pettine in posizione. </li><li> Versare lentamente soluzione di agarosio in tha colata vassoio per evitare la formazione di bolle. Eliminare le bolle utilizzando una punta della pipetta. Attendere il gel raffreddare e solidificare completamente. Una volta solidificato, rimuovere il pettine lentamente per evitare collasso pozzetti mediante aspirazione e rimuovere il nastro dai bordi del vassoio. </li><li> Mettere gel nella casella di gel e riempire l&#39;unità di elettroforesi con 1x TAE-0.1% tampone SDS fino a quando il gel è completamente coperto. </li></ol> 3. Il campione Caricamento e elettroforesi La separazione Nota: Nel nostro laboratorio, usiamo comunemente campioni sia da mouse e gli esseri umani, tra cui il fluido di lavaggio broncoalveolare (BAL; entrambe le specie), lavaggio delle cellule dalle cellule epiteliali bronchiali umane (HBE), e campioni di saliva e di espettorato umani. I campioni possono essere denaturate in 6 M di urea al momento della raccolta o conservati per brevi periodi di tempo (6 ore) con l&#39;aggiunta di inibitore della proteinasi cocktail. <ol><li> Preparare campioni per loading aggiungendo 10% di colorante di caricamento per ciascun campione (es., 30 ml di campione e 3ml di colorante). Omogeneizzare campioni pipettando su e giù. Brevemente spin down tubi a 5.000 giri per raccogliere gocce. </li><li> caricare con cautela uguale volume di campioni nei pozzetti. <ol><li> punte Pre-umidi e / o utilizzare pipette spostamento positivo per facilitare il caricamento dei campioni altamente viscosi. Lentamente aspirato 80-90% della soluzione del campione-dye (per esempio, 27 - 30 ml) per evitare le bolle di pipettamento. </li><li> Lentamente caricare al fondo dei pozzetti e progressivamente spostare la punta della pipetta verso l&#39;alto. Per i campioni che rimangono attaccate alla punta della pipetta, dispensare a 45 ° e allungare dritto sopra pozzo o delicatamente utilizzare il lato del pozzo per rompere il muco filante. Non sovraccaricare i pozzi. </li></ol></li><li> Collegare gli elettrodi dalla scatola gel per il generatore di corrente elettroforesi. Assicurarsi che gli elettrodi siano collegati correttamente (es., Cavo rosso collegato all&#39;uscita positivo del generatore) per consentire negativamente mucine a correre verso il positivo addebitato<html> elettrodo. </li><li> Attivare il gel a 80 Volts per 90 minuti per un singolo gel pettine, o 60 - 75 min per un gel doppio pettine per evitare sovrapposizioni tra le due file. </li><li> SPEGNERE generatore e scollegare gli elettrodi dalla sorgente di alimentazione. </li><li> Rimuovere accuratamente il gel dalla scatola gel con una mano su entrambi i lati del vassoio di colata per garantire il gel rimanga in posizione. </li></ol> 4. Ridurre agarosio gel per un efficiente trasferimento Mucin <ol><li> posizionare con cura il piano di gel in un contenitore di vetro. Lavare il gel con dH 2 O per rimuovere le tracce di tampone TAE-SDS. </li><li> Preparare 1 litro di tampone di 4x SSC aggiungendo 200 ml di 20x SSC in 800 ml di dH 2 O. Rendere 200 ml di 10 mM ditiotreitolo (DTT) soluzione 4x SSC aggiungendo 309 mg di fresco DTT in 200 ml di tampone 4x SSC. </li><li> Immergere il gel con tampone DTT-4x SSC e la copertura. Agitare delicatamente per 20 min (10 rotazioni al minuto). Risciacquare gel con DTT-libera tampone 4x SSC. </li></ol>&quot;&gt; 5. Aspirare Assemblea assorbente e di trasferimento del campione ad una membrana di nitrocellulosa <ol><li> Preparare carta assorbente vuoto per il trasferimento. <ol><li> Asportare accuratamente membrana di nitrocellulosa a dimensioni leggermente più ampie rispetto al gel (es., 10 x 15 cm). </li><li> Wet tappeto poroso con dH 2 O e posizionarlo liscio-side-up nella carta assorbente. </li><li> Wet membrana di nitrocellulosa con tampone SSC 4x DTT-libero e posizionarlo al centro del tappeto porosa. </li><li> Coprire il tappetino poroso con la guarnizione di gomma, lasciando l&#39;apertura della guarnizione precisamente allineato con la membrana di nitrocellulosa. Se mat porosa è ancora visibile, regolare con cura la membrana per garantire l&#39;assenza divario resta tra la guarnizione e la membrana. </li></ol></li><li> posizionare accuratamente il gel di agarosio sopra della membrana. Assicurare forma un gel una tenuta ermetica con la guarnizione ed i pozzetti del gel sono posizionati sulla membrana per il trasferimento. <ol><li> Evitare la formazione di bolle tra la membrana ed il gel. Per minimizzare laformazione di bolle, spruzzare qualche goccia di 4x SSC tampone sulla membrana e far scorrere il gel da cima a fondo per svuotare eventuali bolle formate. Evitare di spostare il gel sulla membrana proteine ​​inizierà a cancellare immediatamente. </li></ol></li><li> coprire con cura il gel con il tampone 4x SSC. </li><li> Inizia trasferimento mucina mediante aspirazione. <ol><li> Accendere carta assorbente vuoto su e impostare la pressione a 40 - 50 mbar. Aggiungere qualche goccia di tampone 4x SSC sul gel ogni 5 - 10 minuti per evitare che il gel si asciughi. trasferimento Run per 90 min. Opzionale: Al termine, segnare i pozzetti con una matita per l&#39;orientamento. </li></ol></li><li> Eliminare il gel e recuperare membrana di nitrocellulosa utilizzando pinzette di plastica sul bordo della membrana. </li></ol> 6. Blocco membrana e rilevamento <ol><li> Risciacquare membrana immediatamente con soluzione salina 1x tampone fosfato (PBS). Non lasciate che la membrana asciugare. </li><li> Immergere la membrana in 50 ml di 3% di latte-PBS tampone di bloccaggio e posto su un rocker a temperatura ambiente per 1 ora. Dopo l&#39;incubazione, scartare il tampone bloccante. </li><li> Immergere membrana 1% soluzione di anticorpo primario latte-PBS. Incubare la membrana nella soluzione di anticorpo primario durante la notte su una sedia a dondolo a 4 ° C. Dopo l&#39;incubazione, scartare la soluzione di anticorpo primario. Diluire gli anticorpi primari ad un rapporto di 1: 2.000 di soluzione di anticorpi al tampone di bloccaggio. Nota: Per i campioni provenienti da topi, usiamo UNC 222 coniglio anti-Muc5b e capra proteina anti-verde fluorescente (GFP). Per i campioni provenienti da esseri umani, usiamo H300 anti-MUC5B e 45M1 anti-MUC5AC anticorpi primari. </li><li> membrana lavata 3 volte con PBS per 10 minuti su un agitatore meccanico. </li><li> Immergere membrana 1% soluzione di anticorpo secondario latte-PBS. Diluire gli anticorpi secondari ad un rapporto di 1: 7.500 di soluzione di anticorpi tampone bloccante e incubare a temperatura ambiente per 1 ora su un agitatore meccanico (ad esempio, 700 anti-coniglio e 680 anti-capra.). Proteggere dalla luce incubando in un co non trasparententainer. Scartare la soluzione di anticorpo secondario dopo 1 ora. </li><li> membrana lavare 3 volte con PBS per 5 min. Risciacquare a membrana con dH 2 O per rimuovere il sale. </li><li> Leggere la membrana su un sistema di fluorescenza a raggi infrarossi secondo le istruzioni del produttore. </li></ol>

<ol>
	<li>Henderson, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis+airway+secretions+exhibit+mucin+hyperconcentration+and+increased+osmotic+pressure.">Cystic fibrosis airway secretions exhibit mucin hyperconcentration and increased osmotic pressure.</a> J Clin Invest. 124 (7), 3047-3060 (2014).</li><li>Preciado, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MUC5B+Is+the+predominant+mucin+glycoprotein+in+chronic+otitis+media+fluid.">MUC5B Is the predominant mucin glycoprotein in chronic otitis media fluid.</a> Pediatr Res. 68 (3), 231-236 (2010).</li><li>Wang, Y. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IgG+in+cervicovaginal+mucus+traps+HSV+and+prevents+vaginal+herpes+infections.">IgG in cervicovaginal mucus traps HSV and prevents vaginal herpes infections.</a> Mucosal Immunol. 7 (5), 1036-1044 (2014).</li><li>Linden, S. K., Sutton, P., Karlsson, N. G., Korolik, V., McGuckin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucins+in+the+mucosal+barrier+to+infection.">Mucins in the mucosal barrier to infection.</a> Mucosal Immunol. 1 (3), 183-197 (2008).</li><li>Thornton, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucus+glycoproteins+from+'normal'+human+tracheobronchial+secretion.">Mucus glycoproteins from 'normal' human tracheobronchial secretion.</a> Biochem J. 265 (1), 179-186 (1990).</li><li>Kesimer, M., Makhov, A. M., Griffith, J. D., Verdugo, P., Sheehan, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unpacking+a+gel-forming+mucin:+a+view+of+MUC5B+organization+after+granular+release.">Unpacking a gel-forming mucin: a view of MUC5B organization after granular release.</a> Am J Physiol Lung Cell Mol Physiol. 298 (1), L15-L22 (2010).</li><li>Kesimer, M., Sheehan, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometric+analysis+of+mucin+core+proteins.">Mass spectrometric analysis of mucin core proteins.</a> Methods Mol Biol. 842, 67-79 (2012).</li><li>van der Post, S., Thomsson, K. A., Hansson, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+enzyme+approach+for+the+characterization+of+glycan+modifications+on+the+C-terminus+of+the+intestinal+MUC2mucin.">Multiple enzyme approach for the characterization of glycan modifications on the C-terminus of the intestinal MUC2mucin.</a> J Proteome Res. 13 (12), 6013-6023 (2014).</li><li>Thornton, D. J., Carlstedt, I., Sheehan, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+glycoproteins+on+nitrocellulose+membranes+and+gels.">Identification of glycoproteins on nitrocellulose membranes and gels.</a> Methods Mol Biol. 32, 119-128 (1994).</li><li>Thornton, D. J., Carlstedt, I., Sheehan, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+glycoproteins+on+nitrocellulose+membranes+and+gels.">Identification of glycoproteins on nitrocellulose membranes and gels.</a> Mol Biotechnol. 5 (2), 171-176 (1996).</li><li>Sheehan, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+characterization+of+the+MUC5AC+mucin:+a+highly+oligomeric+glycoprotein+whether+isolated+from+cell+culture+or+in+vivo+from+respiratory+mucous+secretions.">Physical characterization of the MUC5AC mucin: a highly oligomeric glycoprotein whether isolated from cell culture or in vivo from respiratory mucous secretions.</a> Biochem J. 347 Pt 1, 37-44 (2000).</li><li>Thornton, D. J., Howard, M., Khan, N., Sheehan, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+two+glycoforms+of+the+MUC5B+mucin+in+human+respiratory+mucus.+Evidence+for+a+cysteine-rich+sequence+repeated+within+the+molecule.">Identification of two glycoforms of the MUC5B mucin in human respiratory mucus. Evidence for a cysteine-rich sequence repeated within the molecule.</a> J Biol Chem. 272 (14), 9561-9566 (1997).</li><li>Sheehan, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+molecular+intermediates+in+the+assembly+pathway+of+the+MUC5AC+mucin.">Identification of molecular intermediates in the assembly pathway of the MUC5AC mucin.</a> J Biol Chem. 279 (15), 15698-15705 (2004).</li><li>Ehre, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpressing+mouse+model+demonstrates+the+protective+role+of+Muc5ac+in+the+lungs.">Overexpressing mouse model demonstrates the protective role of Muc5ac in the lungs.</a> Proc Natl Acad Sci U S A. 109 (41), 16528-16533 (2012).</li><li>Livraghi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airway+and+lung+pathology+due+to+mucosal+surface+dehydration+in+{beta}-epithelial+Na++channel-overexpressing+mice:+role+of+TNF-{alpha}+and+IL-4R{alpha}+signaling,+influence+of+neonatal+development,+and+limited+efficacy+of+glucocorticoid+treatment.">Airway and lung pathology due to mucosal surface dehydration in {beta}-epithelial Na+ channel-overexpressing mice: role of TNF-{alpha} and IL-4R{alpha} signaling, influence of neonatal development, and limited efficacy of glucocorticoid treatment.</a> J Immunol. 182 (7), 4357-4367 (2009).</li><li>Martino, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ER+stress+transducer+IRE1beta+is+required+for+airway+epithelial+mucin+production.">The ER stress transducer IRE1beta is required for airway epithelial mucin production.</a> Mucosal Immunol. 6 (3), 639-654 (2013).</li><li>Nguyen, L. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+exposure+to+beta-blockers+attenuates+inflammation+and+mucin+content+in+a+murine+asthma+model.">Chronic exposure to beta-blockers attenuates inflammation and mucin content in a murine asthma model.</a> Am J Respir Cell Mol Biol. 38 (3), 256-262 (2008).</li><li>Papakonstantinou, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COPD+exacerbations+are+associated+with+pro-inflammatory+degradation+of+hyaluronic+acid.">COPD exacerbations are associated with pro-inflammatory degradation of hyaluronic acid.</a> Chest. , (2015).</li><li>Abdullah, L. H., Wolber, C., Kesimer, M., Sheehan, J. K., Davis, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+mucin+secretion+from+human+bronchial+epithelial+cell+primary+cultures.">Studying mucin secretion from human bronchial epithelial cell primary cultures.</a> Methods Mol Biol. 842, 259-277 (2012).</li></ol>

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Il protocollo di mucina Western blotting descritto in questo video combina tecniche convenzionali utilizzate in biologia molecolare per separare e trasferire grandi macromolecole, come il DNA, con tecniche regolari per il rilevamento di proteine, cioè., Immunoblotting. La stessa tecnica può essere applicata per studiare la biologia di glicosaminoglicani complessi, quali la ripartizione di alta peso molecolare dell&#39;acido ialuronico 18. Sebbene questa tecnica potrebbe essere utilizzata in un&#39;...

Mucine sono normalmente prodotti da superfici mucose che rivestono le cavità esposti all&#39;ambiente esterno (ad es., Respiratorio, digestivo, tratto riproduttivo, superficie oculare), così come gli organi interni (ad esempio, pancreas, della cistifellea, ghiandole mammarie). La presenza di queste glicoproteine ​​mantiene l&#39;idratazione superficiale e forma una barriera fisica contro i patogeni. Anche se la produzione di mucina è essenziale per la salute della mucosa, mucina iperconcentrazione e / o muco aberranti può portare a ostruzione del dotto, la colonizzazione batterica e infiammazione cronica, che può causare danni ai tessuti irreversibili. Un simile cascata di eventi sono stati osservati in diverse malattie, per esempio., La fibrosi cistica 1, otite media cronica 2 e l&#39;infezione cervicovaginale 3. Pertanto, è importante comprendere il ruolo di mucine di salute e di malattia e di stabilire protocolli di routine per l&#39;identificazione di proteine. Ad oggi, 19 mucine geni sono stati identificati e codificano per grandi catene polipeptidiche che vanno da 1.200 (ad es., MUC1) a 22.000 (per esempio, MUC16) aminoacidi. La famiglia genica mucina può essere divisa in due sottotipi: le mucine associate alla membrana, coinvolte nella segnalazione cellulare e la superficie schermante e le mucine gelificanti, responsabile delle proprietà viscoelastiche del gel muco. mucine associata alla membrana sono per lo più monomerica e allegare alla superficie delle cellule tramite un dominio di membrana-spanning idrofoba. Al contrario, mucine gelificanti possiedono diversi -come e ricche di cisteina domini fattore di von Willebrand (vWF) che sono essenziali per la formazione di reti polimeriche dinamiche. Grandi glicani sono attaccati alla serina e treonina distribuiti in tutto il apomucin. Questi oligosaccaridi densi O-linked possono contribuire fino al 80% del peso molecolare 4. Intra e inter-molecolari legami disolfuro che collegano monomeri mucina garantire l&#39;integrità del networ gel di mucinaK. Come risultato di glicosilazione pesante e multimerizzazione, mucine sono tra le più grandi molecole nel mondo animale e non possono essere analizzati mediante elettroforesi su gel standard utilizzando convenzionale gel SDS-PAGE poliacrilammide e scale proteine ​​standard. Questi metodi risolvono / proteine ​​separate con pesi molecolari inferiori a 250 kDa mentre monomeri mucina possono raggiungere fino a 2 MDa nel caso di MUC16. Tuttavia, scale di proteine ​​ad alto peso molecolare possono essere utilizzati per studiare piccoli monomeri mucina (es., MUC1). Una varietà di tecniche può essere applicata per studiare dimensioni mucina, conformazione e interazione. Tradizionalmente, caratterizzazione biochimica di mucine si ottiene mucina isolamento tramite isopycnic centrifugazione in gradiente di densità in buffer di denaturazione, seguita da cromatografia dimensione esclusione e immunorilevazione (ad es., Di slot blotting) 5. e / o diffusione della luce multi-angolo dinamico fornire informazioni sullo stato oligomerico di campioni ricchi di mucina &lt;sup&gt; 1. Inoltre, centrifugazione rate-zonale accoppiato con immunolocalizzazione e microscopia elettronica a trasmissione sono comunemente utilizzati per determinare la conformazione macromolecolare di mucine 6. La spettrometria di massa è anche usato per quantificare mucine, rilevare taglio proteolitico e analizzare la composizione oligosaccaride 1,7,8. Tali tecniche sono costose, richiede tempo e spesso richiedono grandi volumi e / o alte concentrazioni di campione. La metodologia descritta, ad esempio., Mucina separazione mediante elettroforesi, è riproducibile, a basso costo e può essere usato in studi di alta throughput per fornire relativa mucina quantificazione e indagare assemblaggio polimero. Tuttavia, questo test richiede ad alta affinità, ad alta specificità anticorpi mucina che potrebbero non essere disponibili per rari mucine (ad es., MUC19) o alcune specie (ad es., Maiale, Ferret). Agarose Western blotting è adatto per risolvere una varietà di campioni ricchi di mucina con concentrazionezioni che vanno da 50 mg / ml (ad es., lavaggi cellulari) a 5 mg / ml (ad es., espettorato). Questo test è stato introdotto nel 1990 ed è stata eseguita solo in pochi laboratori specializzati 9,10. Inizialmente, questa tecnica ha contribuito ad individuare sottopopolazioni di monomeri mucina nelle secrezioni respiratorie dell&#39;uomo 11,12 e confermato il processo di oligomerizzazione in cellule caliciformi, che consiste di formazione dimero nel reticolo endoplasmatico seguita da dimero multimerizzazione nel Golgi 13. Più di recente, la generazione di anticorpi policlonali contro mucine murini facilitato studi su modelli animali di piccole dimensioni (ad es., Mucina deficienti, modelli murini βENaC, OVA-sfidati) e ha aperto un nuovo campo di ricerca per gli studi preclinici composti test farmacologici che mirano a rimuovere il muco dal i polmoni 14-17. Come risultato di un crescente interesse mucina biologia e la generazione di nuovi e più specifici anticorpi mucina, descriviamo herein metodologia per separare mucine mediante elettroforesi su gel di agarosio, trasferimento vuoto nitrocellulosa e rilevazione fluorescente infrarosso bicolore.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tris Base</td>
			<td>Sigma</td>
			<td>T6060-1kg</td>
			<td>1kg</td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>Sigma</td>
			<td>ARK2183-1L</td>
			<td>1L</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>EDS-100g</td>
			<td>100g</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher</td>
			<td>BP229-1</td>
			<td>1L</td>
		</tr>
		<tr>
			<td>Bromophenol Blue</td>
			<td>Sigma</td>
			<td>114391-5g</td>
			<td>5g</td>
		</tr>
		<tr>
			<td>SDS (Sodium Dodecyl Sulfate)</td>
			<td>Sigma</td>
			<td>L6026-50g</td>
			<td>50g</td>
		</tr>
		<tr>
			<td>20X SSC (Sodium Saline Citrate) Buffer</td>
			<td>Promega</td>
			<td>V4261</td>
			<td>1L</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>S271-1</td>
			<td>1kg</td>
		</tr>
		<tr>
			<td>Trisodium Citrate (Na3C6H5O7)</td>
			<td>Sigma</td>
			<td>W302600-1kg</td>
			<td>1kg</td>
		</tr>
		<tr>
			<td>10 mM DTT (dithiothreitol)&#160;</td>
			<td>Sigma</td>
			<td>D0632</td>
			<td>25g</td>
		</tr>
		<tr>
			<td>Milk Powder</td>
			<td>Saco</td>
			<td></td>
			<td>Instant non-fat dry milk</td>
		</tr>
		<tr>
			<td>Dulbecco Phosphate Buffered Saline (D-PBS)</td>
			<td>Gibco lifetechnologies</td>
			<td>14200-025</td>
			<td>500mL</td>
		</tr>
		<tr>
			<td>Anti-GFP Goat Primary Antibody (mouse samples)</td>
			<td>Rockland antibodies and assays</td>
			<td>600-101-215</td>
			<td>1 mg</td>
		</tr>
		<tr>
			<td>UNC 222 Anti-Muc5b Rabbit Primary Antibody (mouse samples)</td>
			<td>UNC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>H300 Anti-MUC5B Primary Antibodies (human samples)</td>
			<td>Santa Cruz</td>
			<td>sc-20119</td>
			<td>200ug</td>
		</tr>
		<tr>
			<td>45M1 Anti-MUC5AC Primary Antibodies (human samples)</td>
			<td>Abcam</td>
			<td>ab3649</td>
			<td>100ug</td>
		</tr>
		<tr>
			<td>Donkey Anti-rabbit 800CW IR Dye</td>
			<td>LI-COR Biosciences</td>
			<td>926-32213</td>
			<td>0.5mg</td>
		</tr>
		<tr>
			<td>Donkey Anti-mouse 680LT IR Dye</td>
			<td>LI-COR Biosciences</td>
			<td>926-68022</td>
			<td>0.5mg</td>
		</tr>
		<tr>
			<td>Electrophoresis Gel Box and Casting Tray</td>
			<td>Owl Seperation Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Power Supply Box</td>
			<td>Biorad</td>
			<td>Model 200/20</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane Blotting Paper</td>
			<td>Amersham&#160;</td>
			<td>10600016&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Paper and Nitrocellulose membrane&#160;</td>
			<td>GE&#160;</td>
			<td>10 439 196</td>
			<td></td>
		</tr>
		<tr>
			<td>Boekel/Appligene Vacuum Blotter 230v</td>
			<td>Expotech USA</td>
			<td>230600-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey Infrared Fluorescence System</td>
			<td>LI-COR Biosciences</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

mucin agarose gel electrophoresis: western blotting for high-molecular-weight glycoproteins

Mucine, le proteine ​​pesantemente glicosilata che rivestono le superfici mucose, si sono evoluti come componente chiave di difesa innata proteggendo l&#39;epitelio contro gli agenti patogeni invasori. Il ruolo principale di queste macromolecole è di facilitare la cattura delle particelle e la clearance favorendo la lubrificazione della mucosa. Durante la sintesi proteica, mucine subiscono intensa O-glicosilazione e multimerizzazione, che aumentano notevolmente la massa e le dimensioni di queste molecole. Queste modificazioni post-traduzionali sono critici per le proprietà viscoelastiche del muco. Come risultato della complessa natura biochimica e biofisica di queste molecole, lavorando con mucine fornisce molte sfide che non possono essere superati mediante metodi di analisi proteica convenzionali. Per esempio, il loro alto peso molecolare impedisce migrazione elettroforetica mediante gel di poliacrilammide regolari e la loro natura appiccicosa provoca l&#39;adesione al tubo sperimentale. Tuttavia, indagando il ruolo di mucine nella salute(Ad es., Mantenendo l&#39;integrità della mucosa) e malattia (ad es., Iperconcentrazione, mucostasis, cancro) ha recentemente guadagnato interesse e mucine sono oggetto di indagine come un obiettivo terapeutico. Una migliore comprensione della produzione e della funzione di macromolecole mucina può portare ad approcci farmaceutici innovativi, ad esempio, inibitori di mucina esocitosi di granuli e / o agenti mucolitici. Pertanto, i protocolli coerenti e affidabili per indagare la biologia mucina sono fondamentali per il progresso scientifico. Qui, descriviamo i metodi convenzionali per separare mucina macromolecole mediante elettroforesi utilizzando un gel, il trasferimento della proteina in membrana di nitrocellulosa, e rilevare il segnale con anticorpi specifici mucina così come lettore di gel a fluorescenza a raggi infrarossi. Queste tecniche sono ampiamente applicabile per determinare mucina quantificazione, multimerizzazione e per testare gli effetti dei composti farmacologici su mucine.

Mostriamo risultati rappresentativi di espressione mucina seguenti elettroforesi su gel di agarosio in BALF dai polmoni di topi (Figura 1). In questo esempio, abbiamo usato il gel per mostrare upregulation di mucina produzione di IL-13 dopo il trattamento del modello di topo Tg-MUC5AC. Il Western blot mostra una rappresentazione visiva di espressione mucina, che può essere utilizzato per l&#39;analisi quantitativa di multimero o l&#39;intensità del segnale di banda mon...

Watch this Scientific Journal Video about Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins at JoVE.com

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

Mucina Agarosio elettroforesi su gel: Western Blotting per glicoproteine ​​ad alto peso molecolare


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

mucin-agarose-gel-electrophoresis-western-blotting-for-high-molecular

Research

JoVE Journal

Biology

25.2K Views.  University of North Carolina at Chapel Hill. Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly. 

Video: Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis DGGE

Okay.Hi, my name is Celeste Peterson and I'm a fellow at Harvard University studying microbial bio film communities. So I am profiling the bacterial communities by looking at their 60 N sequence and how this sequence changes over time in space. And to do that, I'm using DDGE.This is not a normal acry of my well, it needs to be kept at 60 degrees when it's run and the buffer needs to be pumped continuously. So we're gonna warm up the buffer for about an hour until it's at 60 degrees, and then we can run the towel. Now I'm gonna wash this plate with dilute uc solution, peeling around for impurities, andone, light that up.Anything like that can cause regularities. It is important to wipe off all the water. So when I assemble the gel, I'll put that chip on the outside of the gel.So the gel will be in between these two pieces of glass and the chip will be on the outside and it won't affect the gel in between. So now I'm gonna assemble the gel in these clamps and then I squeeze them together and tighten by turning to the right. As with any gel, it's important to make sure that the bottom is flush.To prevent making, we now turn the gel around and put it in here to get sealed with the gasket. So it's important when you do this that the gel, that the plates are actually lined up with the gasket here. So these knobs on the side then go in and you press down to type the seal.So this is a 30 mil syringe. And this part and this part come with the bio red kit and it just on like this. And that's just to get rid of the excess water in the tubes.So put the tubing onto the gel to load the gel. First you put an adapter into the tubing and then a needle look with that small adapter and put it right in the center of the gel just so it's fairly below the bottom glass. Then this wire adapter goes into the long tubing and it's going to connect to equal syringes containing the high and the low density solution.So these are pre-prepared solutions. The blue one, I've added a dye here that just indicates that this is the most denaturing solution and this is the least fitting train solution, the low density one. And they have everything in them.And now I'm gonna to make, gonna be Actually I twice as much the week. And once you've got the tempt, you have about five to seven minutes to fill the gel before the colon rises. And now we fill the syringe juice with the solution.So I'm gonna fill it with about 15 the solution. And then to get rid of any air bubbles, I'll turn it upside down and squeeze gently until that top air bubble is gone. Now this little screw here is gonna go into this groove.This adapter is gonna line up with this clamp here, and it's gonna get C clamped in very tightly. And then this tubing is gonna attach to the syringe. And the same thing is done on the other side with the high density matrix where it's time to deliver.And so I'm gonna be turning this wheel in a steady clockwise motion. And first the blue solution is gonna go out first because that's the the most in nature, and it's gonna form the bottom part of the gel. And then after a little while, the clear solution will start filling in the gel and the Gradient will be formed.One moment is the gently on an angle to And as soon as you finish pouring the gel to wash out your tubes immediately because they will polymerize Going, It's the group there. And then press down. So this little, And on the other side, I'll do the same thing.Okay.Hi, my name is Celeste Peterson and I'm a fellow at Harvard University studying microbial bio communities. So I am profiling the bacterial communities by looking at their 60 F sequence and how the sequence changes over time in space. And to do that, I'm using DDGE.So DGE is Dena gel electrophoresis. And basically the idea is that there's a gradient and the DNA, as it runs down the gradient gets de natured. And so different 16 species will stop at different points along the gradient.And so it ends up being a fingerprint of the different sexiness species in the community. So the goal of my experiment is to see who is in the community and how that's changing over time and space. And I can run the sexiness DDD gel and then I can cut out the bands and identify the species that are represented in each community.So one of the biggest issues when, when doing A DDG gel is to figure out the gradient that's the best. And so that is just something that needs to be played around with and figuring out for a particular community which type of gradient. So the one I'm using today is 35 to 55%urea.One of the big problems that can happen with this technique is that smears come up in in the leans. And I found for that, for whatever reason, one of the best things to do is to run the gels for a really long time and then the smears disappear. Well, today I'm just using it for general bacterial communities, but this can also be used for a specific family of bacteria to see how those change over time, and those tend to be very clean gels.You can also look at mutations in single genes with DDGE.

Denaturazione gel elettroforesi gradiente (DGGE)

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Biologia

Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the relative amount of the protein present in different samples, and analyze the results of co-immunoprecipitation experiments. In this method, a target protein is detected with a specific primary antibody in a given sample of tissue homogenate or extract. Protein separation according to molecular weight is achieved using denaturing SDS-PAGE. After transfer to a membrane, the target protein is probed with a specific primary antibody and detected by chemiluminescence.
Since its first description, the western-blotting technique has undergone several improvements, including pre-cast gels and user-friendly equipment. In our laboratory, we have chosen to use the commercially available NuPAGE electrophoresis system from Invitrogen. It is an innovative neutral pH, discontinuous SDS-PAGE, pre-cast mini-gel system. This system presents several advantages over the traditional Laemmli technique including: i) a longer shelf life of the pre-cast gels ranging from 8 months to 1 year; ii) a broad separation range of molecular weights from 1 to 400 kDa depending of the type of gel used; and iii) greater versatility (range of acrylamide percentage, the type of gel, and the ionic composition of the running buffer).
The procedure described in this video article utilizes the Bis-Tris discontinuous buffer system with 4-12% Bis-Tris gradient gels and MES running buffer, as an illustration of how to perform a western-blot using the Invitrogen NuPAGE electrophoresis system. In our laboratory, we have obtained good and reproducible results for various biochemical applications using this western-blotting method.

This technical article describes a standard western-blotting procedure using the commercially available NuPAGE electrophoresis Mini-Gel system from Invitrogen.

Western Blotting Utilizzando il Invitrogen NuPage Novex Bis Tris MiniGels

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the ...

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

Valutare motilità delle cellule staminali neurali L&#39;utilizzo di un gel a base di Agarose dispositivo a microfluidi

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

Lo screening per aggregazione amiloide di semi-denaturazione detergente-Agarosio elettroforesi su gel

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method1. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 &deg;C during the gel run allows for the separation of unstructured DNA or RNA molecules.
In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.
In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol 1,2.

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.

Denaturazione Urea elettroforesi su gel poliacrilammide (PAG Urea)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

Estrazione di DNA ad alta peso molecolare genomico da terreni e sedimenti

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

Blu nativi poliacrilammide gel elettroforesi (BN-PAGE) per l&#39;analisi di complessi multiproteici da lisati cellulari

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types and is widely believed to be directly associated with the development of cancer in humans. DNA damage, such as that induced by UV irradiation, severely impairs the ability of replication to duplicate the genomic template accurately. A number of gene products have been identified that are required when replication encounters DNA lesions in the template. However, a remaining challenge has been to determine how these proteins process lesions during replication in vivo. Using Escherichia coli as a model system, we describe a procedure in which two-dimensional agarose-gel analysis can be used to identify the structural intermediates that arise on replicating plasmids in vivo following UV-induced DNA damage. This procedure has been used to demonstrate that replication forks blocked by UV-induced damage undergo a transient reversal that is stabilized by RecA and several gene products associated with the RecF pathway. The technique demonstrates that these replication intermediates are maintained until a time that correlates with the removal of the lesions by nucleotide excision repair and replication resumes.

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

We present a procedure by which two-dimensional agarose-gel analysis can be used to identify the structure of replication intermediates that occur following UV irradiation.

Visualizzazione dei raggi UV-indotta Intermedi replica in E. coli Utilizzando bidimensionale agarosio-gel Analisi

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types ...

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

Western Blotting: la preparazione dei campioni di rilevamento

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

Estrazione di DNA ad alta peso molecolare da Mats microbica

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

Mucina Agarosio elettroforesi su gel: Western Blotting per glicoproteine ad alto peso molecolare

Riepilogo

Esplora Altri Video

Capitoli in questo video

Denaturazione gel elettroforesi gradiente (DGGE)

Western Blotting Utilizzando il Invitrogen NuPage Novex Bis Tris MiniGels

Valutare motilità delle cellule staminali neurali L'utilizzo di un gel a base di Agarose dispositivo a microfluidi

Lo screening per aggregazione amiloide di semi-denaturazione detergente-Agarosio elettroforesi su gel

Denaturazione Urea elettroforesi su gel poliacrilammide (PAG Urea)

Estrazione di DNA ad alta peso molecolare genomico da terreni e sedimenti

Blu nativi poliacrilammide gel elettroforesi (BN-PAGE) per l'analisi di complessi multiproteici da lisati cellulari

Visualizzazione dei raggi UV-indotta Intermedi replica in E. coli Utilizzando bidimensionale agarosio-gel Analisi

Western Blotting: la preparazione dei campioni di rilevamento

Estrazione di DNA ad alta peso molecolare da Mats microbica

Mucina Agarosio elettroforesi su gel: Western Blotting per glicoproteine ​​ad alto peso molecolare

Riepilogo

Esplora Altri Video

Capitoli in questo video

Denaturazione gel elettroforesi gradiente (DGGE)

Western Blotting Utilizzando il Invitrogen NuPage Novex Bis Tris MiniGels

Valutare motilità delle cellule staminali neurali L'utilizzo di un gel a base di Agarose dispositivo a microfluidi

Lo screening per aggregazione amiloide di semi-denaturazione detergente-Agarosio elettroforesi su gel

Denaturazione Urea elettroforesi su gel poliacrilammide (PAG Urea)

Estrazione di DNA ad alta peso molecolare genomico da terreni e sedimenti

Blu nativi poliacrilammide gel elettroforesi (BN-PAGE) per l'analisi di complessi multiproteici da lisati cellulari

Visualizzazione dei raggi UV-indotta Intermedi replica in E. coli Utilizzando bidimensionale agarosio-gel Analisi

Western Blotting: la preparazione dei campioni di rilevamento

Estrazione di DNA ad alta peso molecolare da Mats microbica

Mucina Agarosio elettroforesi su gel: Western Blotting per glicoproteine ad alto peso molecolare