Vorremmo ringrazia per il sostegno finanziario: BBSRC Istituto strategico Programma di finanziamento - CF &amp; TMW; Finanziamento BBSRC Oriente Bio DTP - LG; Il Darwin fiducia di Edimburgo - MLH. Vorremmo anche ringraziare il dottor Barry McColl il permesso di includere l&#39;ottimizzazione TREM2 in questo manoscritto.

<ol>
	<li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Biotechnology. 1992 (24), 145-149 (1979).</li><li>Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protein Protocols on CD-ROM. , (1998).</li><li>Gallagher, S., Winston, S. E., Fuller, S. A., Hurrell, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoblotting+and+immunodetection.">Immunoblotting and immunodetection.</a> Curr Protoc Mol Biol. Chapter. 10, 10 (2008).</li><li>Eaton, S. L., 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=Total+protein+analysis+as+a+reliable+loading+control+for+quantitative+fluorescent+Western+blotting.">Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting.</a> PLoS One. 8 (8), 72457 (2013).</li><li>Suzuki, O., Koura, M., Noguchi, Y., Uchio-Yamada, K., Matsuda, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+sample+mixtures+for+standard+curve+creation+in+quantitative+Western+blots.">Use of sample mixtures for standard curve creation in quantitative Western blots.</a> Exp. Anim. 60, 193-196 (2011).</li><li>Colella, A. 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=Comparison+of+Stain-Free+gels+with+traditional+immunoblot+loading+control+methodology.">Comparison of Stain-Free gels with traditional immunoblot loading control methodology.</a> Anal Biochem. 430 (2), 108-110 (2012).</li><li>Zellner, M., 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=Fluorescence-based+Western+blotting+for+quantitation+of+protein+biomarkers+in+clinical+samples.">Fluorescence-based Western blotting for quantitation of protein biomarkers in clinical samples.</a> Electrophoresis. 29, 3621-3627 (2008).</li><li>Kielar, 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=Molecular+correlates+of+axonal+and+synaptic+pathology+in+mouse+models+of+Batten+disease.">Molecular correlates of axonal and synaptic pathology in mouse models of Batten disease.</a> Hum Mol Genet. 18, 4066-4080 (2009).</li><li>Mutsaers, C. A., Lamont, D. J., Hunter, G., Wishart, T. M., Gillingwater, 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=Label-free+proteomics+identifies+Calreticulin+and+GRP75/Mortalin+as+peripherally+accessible+protein+biomarkers+for+spinal+muscular+atrophy.">Label-free proteomics identifies Calreticulin and GRP75/Mortalin as peripherally accessible protein biomarkers for spinal muscular atrophy.</a> Genome Med. 5 (10), 95 (2013).</li><li>Wishart, T. M., 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=Dysregulation+of+ubiquitin+homeostasis+and+&#946;-catenin+signaling+promote+spinal+muscular+atrophy.">Dysregulation of ubiquitin homeostasis and &#946;-catenin signaling promote spinal muscular atrophy.</a> J Clin Invest. 124 (4), 1821-1834 .</li><li>Mathews, S. T., Plaisance, E. P., Kim, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+systems+for+westerns:+chemiluminescence+vs.+infrared+detection.">Imaging systems for westerns: chemiluminescence vs. infrared detection.</a> Methods Mol Biol. 536, 499-513 (2009).</li><li>Hawes, J. J., Brunzell, D. H., Wynick, D., Zachariou, V., Picciotto, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GalR1,+but+not+GalR2+or+GalR3,+levels+are+regulated+by+galanin+signalling+in+the+locus+coeruleus+through+a+cyclic+AMP-dependent+mechanism.">GalR1, but not GalR2 or GalR3, levels are regulated by galanin signalling in the locus coeruleus through a cyclic AMP-dependent mechanism.</a> J Neurochem. 93 (5), 1168-1176 (2006).</li><li>Bond, D., Primrose, D. A., Foley, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+evaluation+of+signaling+events+in+Drosophila+S2+cells.">Quantitative evaluation of signaling events in Drosophila S2 cells.</a> Biol Proceed Online. 10 (1), 20-28 (2008).</li><li>Wishart, T. M., 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=Differential+proteomics+analysis+of+synaptic+proteins+identifies+potential+cellular+targets+and+protein+mediators+of+synaptic+neuroprotection+conferred+by+the+slow+Wallerian+degeneration+(Wlds)+gene.">Differential proteomics analysis of synaptic proteins identifies potential cellular targets and protein mediators of synaptic neuroprotection conferred by the slow Wallerian degeneration (Wlds) gene.</a> Mol Cell Proteomics. 6, 1318-1330 (2007).</li><li>Silva, J. M., McMahon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fastest+western+in+town:+a+contemporary+twist+on+the+classic+western+blot+analysis.">The fastest western in town: a contemporary twist on the classic western blot analysis.</a> J. Vis. Exp. (84), 51149 (2014).</li></ol>

The authors have nothing to disclose.

Adeguata considerazione e pianificazione è essenziale prima di ogni esperimento e possono infine determinare il successo della tecnica utilizzata. I progressi nella rilevazione della proteina utilizzando WB può presentare una pletora di potenziali ostacoli quando si cerca di scegliere gli anticorpi del caso, di trasferimento e visualizzazione metodi da utilizzare. Per fortuna, con una lista di controllo attento e misure di controllo adeguate QFWB può essere utilizzato di routine per determinare la presenza di proteine ​​e differenze sempre più sottili di espressione tra i campioni. Questo protocollo fornisce una guida completa a fluorescenza western blotting quantitativi così come un paio di strategie di risoluzione dei problemi al fine di evitare e / o superare alcuni dei molti problemi comuni ad esso associati. Le fasi critiche impiegate per mantenere la sensibilità ed ottenere veramente misurazioni quantificabili e comparabili includono: 1) estrazione delle proteine ​​robusta da campioni di tessuto; 2) preparazione del campione; 3) accurata loadin proteineg determinata mediante analisi di proteine ​​totali; 4) trasferimento ottimale di proteine ​​che utilizzano I-Blot; 5) preparazione di anticorpi primari e secondari in tampone di bloccaggio contenente 0,1% Tween20 e 6) corretta visualizzazione e l&#39;analisi utilizzando una termocamera a raggi infrarossi e il software associato. Rilevamento a fluorescenza a raggi infrarossi è veramente quantitativa e fornisce una maggiore sensibilità rispetto alle più tradizionali tecniche di rilevazione ECL 3,11. Questo sistema di rilevamento è multi sfaccettato, e come tale non è limitato a QFWB. Questo sistema è in grado di generare immagini di etichettatura immunoistologico a bassa potenza consentendo la visualizzazione e la quantificazione delle intere sezioni di tessuto 12. Questa è una zona di sviluppo potenziale futuro in termini di risoluzione che potrebbe vedere immagini molto rosso rivaleggiare cattura immunofluorescenza convenzionale con microscopi convenzionali in termini di valutazione quantitativa. Tuttavia, con una maggiore sensibilità ai cambiamenti sottili protein espressione è fondamentale per garantire la variabilità è ridotta al minimo e le misure di controllo sono rigorosi con i protocolli robusti. Questo inizia con l&#39;estrazione delle proteine ​​rigorosa dal campione di tessuto seguita dalla produzione di proteine ​​macchiato totale gel da assicurare che il caricamento del campione è uniforme, l&#39;ottimizzazione di anticorpi primari e secondari al fine di determinare se il rilevamento è reale e testare le linee guida del produttore per quanto riguarda il trasferimento volte per ottenere un efficace trasferimento delle proteine. Tuttavia, anche quando sono ottimizzate le condizioni per WB, ci possono ancora problemi associati alla gestione di western che potrebbero non essere stati completamente esplorati qui. Questi includono ma non sono limitati a fattori tra cui proteine ​​solubilizzazione e la scelta di tampone di estrazione. Alcuni buffer possono interferire con saggi di concentrazione di proteine, e alcuni tessuti sono particolarmente difficili da solubilizzare, richiedono tecniche più robuste come l&#39;uso di macerazione automatizzatoting contenitori sigillati quali tubi M insieme ad un dissociatore Mac. Inoltre, semplici misure di controllo per la conservazione di materiale estratto e non estratti a -80 ° C può essere la differenza tra ottenere etichettatura ottimale subito dopo l&#39;estrazione ed aventi scarsi risultati settimane più tardi. Metodi QFWB moderni hanno dimostrato di essere più sensibile per l&#39;acquisizione di sottili differenze di espressione proteica e sono più versatili permettendo simultanea doppia etichettatura 3 rispetto alle tecniche più vecchi come ECL. E &#39;fondamentale che i protocolli di Western blotting sono robusti e facilmente ripetibile per la quantificazione accurata e l&#39;analisi statistica. Questo protocollo è sensibile e abbastanza robusto per essere usata come metodo per la rilevazione di proteine ​​attraverso una varietà di campioni di tessuto e specie 3 diversi e consente la quantificazione delle proteine ​​poco abbondanti e alti nella stessa QFWB riducendo così l&#39;utilizzo di consumo nonché volta per esperimento 1. 1 Inoltre, la maggiore sensibilità di questa tecnica permette la validazione di sempre più popolare - studi omiche 9,14 tuttavia la precisione è fondamentale e l&#39;inclusione di appropriate misure di controllo devono essere rispettate evitando in tal modo l&#39;acquisizione dei dati errati.

Western blotting (WB) è una tecnica analitica originariamente sviluppato alla fine del 1970 per determinare la presenza o l&#39;assenza di una proteina di interesse in un campione biologico complesso, ad esempio un tessuto omogenato 1. Comunemente denominato immunoblot proteine, a causa dell&#39;interazione antigene-anticorpo chiave, la metodologia consiste di 5 fasi distinte: 1) separazione elettroforetica delle proteine ​​da loro punto isoelettrico; 2) trasferimento ad un nitrocellulosa o di polivinilidene (PVDF) membrana; 3) etichettatura utilizzando un anticorpo primario specifico per la proteina di interesse; 4) incubazione con un anticorpo secondario diretto contro l&#39;anticorpo primario; e 5) visualizzazione. Metodologia di visualizzazione si è evoluto nel tempo per migliorare la sicurezza e la sensibilità. Alcuni dei primi WB sono state effettuate utilizzando radiofoniche tag etichettati che poi progredita al chemilluminescent più diffuso (ECL) metodi colorimetrici e poi. Radioactivity è stato marcato direttamente su sonde di antigeni specifici, mentre metodologie colorimetriche e ECL utilizzano una tecnica di marcatura indiretto con un giornalista di enzimi come la fosfatasi alcalina o perossidasi streptavidina rafano (HRP) 2. L&#39;intensità di etichettatura del prodotto cromogeno o luminescente è misurata utilizzando densitometria per cui la potenza del segnale, forte o debole, indica più o meno la presenza della proteina di interesse nel campione. ECL è il più sensibile e quindi favorita la metodologia 2, ma tutti e 3 i metodi sono stati inizialmente sviluppati in un film x-ray con più sofisticate tecniche di imaging digitale successivamente stabiliti 3. L&#39;avanzamento di imaging digitale di WB consentito non solo un ricercatore per determinare la presenza o l&#39;assenza della loro proteina di interesse, ma anche consentito una deduzione al livello di espressione di una proteina selezionata quando confrontato con altri campioni e quindi può essere indicato come &quot;semi-quantitativa &amp; #8221 ;. Recentemente, una tecnologia western blotting veramente quantitativa e più sensibile è stato sviluppato per cui il livello di fluorescenza misurata è direttamente proporzionale alla quantità e l&#39;espressione di una singola proteina all&#39;interno di un campione: quantitativa fluorescente Western blotting (QFWB). Confrontando QFWB all&#39;etichettatura ECL, l&#39;utilizzo di un anticorpo secondario fluorescente genera un profilo rivelazione lineare 4. Questo è in contrasto con le tecniche ECL, dove la linearità del segnale si verifica in genere con un sacco di proteine ​​basse inferiori a 5 mg e la saturazione del segnale direttamente legati alla espressione della proteina, cioè, con geni housekeeping ubiquitariamente espressi 5,6. Questa disparità è probabilmente causato da un maggior numero di siti di legame disponibili per un substrato avidina ECL lega ad uno secondario biotinilato, risultando in una maggiore probabilità di potenziale saturazione del segnale. Questo è uno dei motivi principali per ECL base di immunoblotting essere indicato come only &quot;semi-quantitativa&quot; 7. Il punto di saturazione del segnale è di fondamentale importanza quando si misura sottili differenze nei livelli di espressione e può portare a misurazioni imprecise. Negli ultimi anni l&#39;avvento di diffuse tecniche di proteomica in dettaglio sempre maggiore sensibilità e l&#39;identificazione dei differenziali di espressione sottili ha comportato un ricorso sempre maggiore a western blotting veramente quantitativo per esperimenti di validazione 8,9. L&#39;applicazione della metodologia sensibile, robusto e veramente quantitativa è quindi cruciale. Molte metodologie di Western blotting &quot;ottimizzate&quot; sono stati utilizzati da laboratori indipendenti, che spesso risultano difficili da creare o replicare a causa di lievi modifiche metodologiche che potrebbero non essere evidenti nei protocolli formali documentate. Si tratta di un protocollo stabilito e robusto per QFWB e inoltre fornisce preziose strategie per la risoluzione dei problemi comuni that possono sorgere in fase di attuazione. Questo protocollo è stato originariamente ottimizzato per l&#39;utilizzo con omogenati di cervello di topo, ma da allora è stato effettivamente utilizzato in una vasta gamma di campioni di tessuto e specie 4,9,10. Variazioni di protocollo potenziali necessari per risolvere i problemi specifici sono inclusi.

<table border="0" cellpadding="0" cellspacing="0" width="1024">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:427px;">Name of Material/ Equipment</td>
			<td style="width:255px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:224px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">RIPA Buffer</td>
			<td style="width:255px;">Fisher Scientific UK Ltd</td>
			<td>10230544</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">M Tubes</td>
			<td style="width:255px;">Miltenyi Biotec Inc.</td>
			<td>130-093-236</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:427px;">iBlot Transfer Stack, PVDF Regular</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td>IB401001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">MagicMark XP Western Protein Standard (20-220 kDa)</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td>LC5602</td>
			<td>Use in gel 1 for Western blotting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">SeeBlue Pre-stained protein standard</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td>LC5625&#160;</td>
			<td>Use in gel 2 Total protein stained gel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">NuPAge LDS Sample buffer 4X</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td>NP0007</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:427px;">NuPAGE MES SDS Running Buffer (for Bis-Tris Gels only) (20X)</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td>NP0002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">NuPAGE Novex 4-12% Bis-Tris Gel 1.0 mm, 12 well</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td>NP0322BOX</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:427px;">PHOSPHATE BUFFERED SALINE TABLET,*TRU-ME, PHOSPHATE BUFFERED SALINE TABLET</td>
			<td style="width:255px;">Sigma-Aldrich, UK</td>
			<td>P4417-100TAB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Micro BCA, Protein Assay Kit</td>
			<td style="width:255px;">Fisher Scientific UK Ltd</td>
			<td>10249133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Odyssey&#160; blocking buffer</td>
			<td style="width:255px;">Li-Cor Biosciences</td>
			<td>P/N 927-40000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">IRDye 680RD Goat anti-Rabbit IgG (H+L), 0.5 mg</td>
			<td style="width:255px;">Li-Cor Biosciences</td>
			<td style="width:119px;">926-68071</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">IRDye 680RD Donkey anti-Mouse IgG (H+L), 0.5 mg&#160;</td>
			<td style="width:255px;">Li-Cor Biosciences</td>
			<td style="width:119px;">926-68072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">IRDye 800CW Goat anti-Rabbit IgG (H + L), 0.5mg</td>
			<td style="width:255px;">Li-Cor Biosciences</td>
			<td style="width:119px;">926-32211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">IRDye 800CW Donkey anti-Goat IgG (H + L), 0.5mg</td>
			<td style="width:255px;">Li-Cor Biosciences</td>
			<td style="width:119px;">926-32214</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">ODYSSEY CL Infra-red imager</td>
			<td style="width:255px;">Li-Cor Biosciences</td>
			<td style="width:119px;"></td>
			<td>Call for quotation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">iBlot 7-Minute Blotting System</td>
			<td style="width:255px;">Life technologies, UK</td>
			<td style="width:119px;"></td>
			<td>This model is no longer in production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">InstantBlue Protein stain</td>
			<td style="width:255px;">Expedeon, UK</td>
			<td>ISB1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Revitablot western blot stripping buffer&#160;</td>
			<td>Rockland Immunochemicals Inc.</td>
			<td>MB-085-0050</td>
			<td></td>
		</tr>
	</tbody>
</table>

a guide to modern quantitative fluorescent western blotting with troubleshooting strategies

Fine del 1970 ha visto il primo uso segnalata pubblicamente del Western Blot, una tecnica per valutare la presenza e l&#39;abbondanza relativa di proteine ​​specifiche all&#39;interno di campioni biologici complessi. Da allora, la metodologia western blotting è diventata una componente comune dei biologi molecolari repertorio sperimentale. Una ricerca sommaria di PubMed usando il termine &quot;Western Blot&quot; suggerisce che in eccesso di 220.000 manoscritti pubblicati hanno fatto uso di questa tecnica per l&#39;anno 2014. È importante sottolineare che, negli ultimi dieci anni hanno visto progressi di imaging tecnici accoppiati con lo sviluppo etichette fluorescenti di sensibili che hanno migliorato la sensibilità e diedero ancora più gamme di rilevamento lineare. Il risultato è una fluorescenza ora veramente quantificabile basato Western Blot (QFWB) che permette ai biologi di effettuare analisi di espressione comparativa con maggiore sensibilità e precisione rispetto al passato. Molti &quot;ottimizzato&quot; western blottingesistono metodologie e sono utilizzati in diversi laboratori. Questi spesso risultare difficile a causa della necessità di modifiche procedurali sottili, ma senza documenti. Questo protocollo fornisce una descrizione completa di un metodo QFWB consolidata e robusto, completo di strategie di risoluzione dei problemi.

Come sensibilità QFWB e la gamma lineare di rilevamento è superiore a rilevamento convenzionale ECL, ci sono una serie di misure di controllo che sono fondamentali per garantire che i dati accurati vengono raccolti, favorendo in tal modo l&#39;interpretazione efficace. In primo luogo, l&#39;inclusione di campioni di controllo positivi come mostrato in Figura 1. In secondo luogo, l&#39;ottimizzazione di trasferimento per garantire circolazione equivalenti di proteine ​​alto e basso peso molecolare dal gel alla membrana come mostrato in Figura 2. In terzo luogo, l&#39;ottimizzazione di anticorpi, in particolare secondaria anticorpi la cui ottimizzazione è spesso trascurato, ma che può produrre non specifico banding in grado di interferire con la corretta interpretazione di proteine ​​(s) di interesse. Vedere Figura 3. In quarto luogo, esso può anche essere il caso che, quando una proteina appare rilevabile ma dovrebbe essere presente, questo può anche essere un problema di anticorpo secondario che può essere corretta tramite una semplice rais secondaricato in un ospite di specie diversa. Vedere Figura 4. In quinto luogo, l&#39;etichettatura proteine ​​totali e l&#39;analisi è un metodo molto più robusto e quantificabile in confronto all&#39;uso di proteine ​​singola tradizionale (s) che sono ubiquitariamente espresso per gli standard di riferimento interno 3. Molte di queste singole proteine ​​sono stati trovati per essere espressi in modo differenziale in modelli di malattie neurodegenerative e tra campioni di tessuto differenti e l&#39;uniformità di espressione può variare all&#39;interno dello stesso tessuto 3. Pertanto, la produzione di un gel di controllo carico confermerà l&#39;uniformità del carico del campione quando combinato con un&#39;analisi proteine ​​totali confrontando e quantificare il carico di proteine ​​in ogni corsia a vari intervalli di pesi molecolari misurati contro ogni campione per indicare errore standard come mostrato in figura 6 . È importante sottolineare che tutte queste tecniche e controlli risoluzione dei problemi sono solo efficace come la sensibilità e coerenza dell&#39;analisi tools applicati dal gestore (Figura 5). Infine questa tecnica si presta a stripping e ri-probing delle membrane con maggiore flessibilità rispetto ECL causa di fattori tra cui, ma non limitato ad una maggiore sensibilità, ridotta sfondo, rilevamento doppio colore e stabilità della membrana in condizioni di stoccaggio a lungo termine. Vedere la Figura 7.

Watch this Scientific Journal Video about A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies at JoVE.com

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

Una guida al moderno Quantitative Fluorescent Western Blotting con strategie di risoluzione dei problemi

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

a-guide-to-modern-quantitative-fluorescent-western-blotting-with

Research

JoVE Journal

Biology

50.7K Views.  University of Edinburgh. The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

Video: A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

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 ...

Ricerca

Biologia

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

Un approccio pratico alla mappatura genetica Destino inducibile: una guida visiva a Mark e Pista Cellule In Vivo</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

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 ...

Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of microbiology was transformed with the advent of Anton van Leeuwenhoek&#39;s microscope, which allowed scientists to visualize prokaryotes for the first time. The development of the polymerase chain reaction (PCR) is one of those innovations that changed the course of molecular science with its impact spanning countless subdisciplines in biology. The theoretical process was outlined by Keppe and coworkers in 1971; however, it was another 14 years until the complete PCR procedure was described and experimentally applied by Kary Mullis while at Cetus Corporation in 1985. Automation and refinement of this technique progressed with the introduction of a thermal stable DNA polymerase from the bacterium Thermus aquaticus, consequently the name Taq DNA polymerase.
PCR is a powerful amplification technique that can generate an ample supply of a specific segment of DNA (i.e., an amplicon) from only a small amount of starting material (i.e., DNA template or target sequence). While straightforward and generally trouble-free, there are pitfalls that complicate the reaction producing spurious results. When PCR fails it can lead to many non-specific DNA products of varying sizes that appear as a ladder or smear of bands on agarose gels. Sometimes no products form at all. Another potential problem occurs when mutations are unintentionally introduced in the amplicons, resulting in a heterogeneous population of PCR products. PCR failures can become frustrating unless patience and careful troubleshooting are employed to sort out and solve the problem(s). This protocol outlines the basic principles of PCR, provides a methodology that will result in amplification of most target sequences, and presents strategies for optimizing a reaction. By following this PCR guide, students should be able to: 
 
&#9679; Set up reactions and thermal cycling conditions for a conventional PCR experiment 
&#9679; Understand the function of various reaction components and their overall effect on a PCR experiment 
&#9679; Design and optimize a PCR experiment for any DNA template 
&#9679; Troubleshoot failed PCR experiments

PCR has emerged as a common technique in many molecular biology laboratories. Provided here is a quick guide to several conventional PCR protocols. Because each reaction is a unique experiment, optimal conditions required to generate a product vary. Understanding the variables in a reaction will greatly enhance troubleshooting efficiency, thereby increasing the chance to obtain the desired result.

Polymerase Chain Reaction: protocollo Basic Plus Strategies Risoluzione dei problemi e ottimizzazione

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of ...

V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black box&#34; because an experimentalist does not know if it has been performed successfully until the last of several steps. Moreover, the quality of western blot data is sometimes challenged due to a lack of effective quality control tools in place throughout the western blotting process. Here we describe the V3 western workflow, which applies stain-free technology to address the major concerns associated with the traditional western blot protocol. This workflow allows researchers: 1) to run a gel in about 20-30 min; 2) to visualize sample separation quality within 5 min after the gel run; 3) to transfer proteins in 3-10 min; 4) to verify transfer efficiency quantitatively; and most importantly 5) to validate changes in the level of the protein of interest using total protein loading control. This novel approach eliminates the need of stripping and reprobing the blot for housekeeping proteins such as &#946;-actin, &#946;-tubulin, GAPDH, etc. The V3 stain-free workflow makes the western blot process faster, transparent, more quantitative&#160;and reliable.

V3 workflow is a western blot procedure using stain-free gels. The stain-free technology allows researchers to visualize protein separation quality, to verify the transfer efficiency, and most importantly, to validate the change in the protein of interest using total protein quantification as a reliable loading control.

Flusso di lavoro senza macchie V3 per un controllo pratico, conveniente e affidabile del carico totale delle proteine nell'blotting occidentale

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black ...

A Practical Guide to Phylogenetics for Nonexperts

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this topic and so it presents inherent problems. Here we compile a practical introduction to phylogenetics for nonexperts.&#160;We outline in a step-by-step manner, a pipeline for generating reliable phylogenies from gene sequence datasets. We begin with a user-guide for similarity search tools via online interfaces as well as local executables. Next, we explore programs for generating multiple sequence alignments followed by protocols for using software to determine best-fit models of evolution. We then outline protocols for reconstructing phylogenetic relationships via maximum likelihood and Bayesian criteria&#160;and finally describe tools for visualizing phylogenetic trees. While this is not by any means an exhaustive description of phylogenetic approaches, it does provide the reader with practical starting information on key software applications commonly utilized by phylogeneticists. The vision for this article would be that it could serve as a practical training tool for researchers embarking on phylogenetic studies&#160;and also serve as an educational resource that could be incorporated into a classroom or teaching-lab.

Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis.&#160;

Una guida pratica per Phylogenetics per i non esperti

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.

This protocol explores the latest advancements in performing Western blot analyses.&#160;These novel modifications employ a Bis-Tris gel system with a 35&#160;min electrophoresis run time, a 7&#160;min dry blotting transfer system, and infrared fluorescent protein detection and imaging that generates higher resolution, quality, sensitivity, and improved accuracy of Western data.

Il più veloce occidentale in città: un tocco contemporaneo alla classica analisi Western Blot

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of ...

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.
In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.

Floral-Dip Transformation of Flax Linum usitatissimum to Generate Transgenic Progenies with a High Transformation Rate

Here, we present a protocol to transform flax using Agrobacterium-mediated plant transformation via floral-dip. This protocol is simple to perform and inexpensive, yet yields a higher transformation rate than the current available methods for flax transformation.

Trasformazione Floral-Dip di lino (<em&gt; Linum usitatissimum</em&gt;) Per generare transgenici progenie con una trasformazione Tasso alta

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded &#180;indefinitely&#180;. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.

Barcoding genetica con proteine ​​fluorescenti per applicazioni multiplexati

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.

Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

Sviluppo di un Quantitative ricombinasi Polymerase Amplificazione Assay con un controllo positivo interno

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to ...