Nous tenons à remercier pour le soutien financier qui suit: BBSRC Institut stratégique de financement des programmes - CF &amp; TMW; Financement BBSRC Est Bio DTP - LG; Le Darwin Trust of Edinburgh - MLH. Nous tenons également à remercier le Dr Barry McColl pour permission d&#39;inclure l&#39;optimisation TREM2 dans ce manuscrit.

<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.

Une attention particulière et de la planification est essentielle avant toute expérience et peuvent en fin de compte déterminer le succès de la technique utilisée. Les progrès dans la détection de la protéine à l&#39;aide de la Banque mondiale peut présenter une pléthore d&#39;obstacles potentiels en essayant de choisir les anticorps, de transfert et de visualisation des méthodes appropriées à utiliser. Heureusement, en utilisant une liste de contrôle attentif et des mesures de contrôle appropriées QFWB peut être utilisée en routine pour déterminer la présence de la protéine et des différences d&#39;expression de plus en plus subtiles entre les échantillons. Ce protocole fournit un guide complet fluorescent western blot quantitatif ainsi que quelques stratégies de dépannage pour éviter et / ou de surmonter quelques-uns des nombreux écueils les plus courants associés. Les étapes essentielles utilisées pour maintenir la sensibilité et obtenir vraiment des mesures quantifiables et comparables comprennent: 1) l&#39;extraction des protéines robuste à partir d&#39;échantillons de tissus; 2) la préparation des échantillons; 3) précise loadin de protéinesg déterminée par l&#39;analyse des protéines totales; 4) un transfert optimal de protéines en utilisant I-Blot; 5) la préparation d&#39;anticorps primaires et secondaires dans le tampon de blocage contenant 0,1% de Tween 20, et 6) visualisation correcte et analyse à l&#39;aide d&#39;un imageur infrarouge et les logiciels associés. Détection fluorescente infrarouge est vraiment quantitative et offre une plus grande sensibilité par rapport à d&#39;autres techniques de détection ECL traditionnelles 3,11. Ce système de détection est à plusieurs facettes, et en tant que tel ne se limite pas à QFWB. Ce système est capable de formation d&#39;image de marquage immunohistochimique à faible puissance permettant la visualisation et la quantification des coupes de tissus entiers 12. Ceci est une zone de développement potentiel futur en termes de résolution qui pourrait voir l&#39;imagerie rouge lointain rivalisant avec capture d&#39;immunofluorescence conventionnelle avec des microscopes conventionnels en termes d&#39;évaluation quantitative. Cependant, avec une plus grande sensibilité à des changements subtils dans protein expression, il est essentiel de veiller à la variabilité est réduite au minimum et les mesures de contrôle sont strictes avec des protocoles robustes. Cela commence avec l&#39;extraction des protéines rigoureuse de l&#39;échantillon de tissu suivie par la production de protéines colorées au total gels à fournir l&#39;assurance que le chargement des échantillons est uniforme, optimisation des anticorps primaires et secondaires afin de déterminer si la détection est réel et tester les directives du fabricant en ce qui concerne le transfert fois pour obtenir le transfert de la protéine efficace. Néanmoins, même lorsque les conditions de la Banque mondiale sont optimisés, il peut y avoir encore des problèmes associés à la gestion des westerns qui peuvent ne pas avoir été pleinement explorées ici. Ceux-ci comprennent, sans s&#39;y limiter, les facteurs, y compris la solubilisation de la protéine et le choix de tampon d&#39;extraction. Des tampons peuvent interférer avec des dosages de la concentration de protéine, et certains tissus sont particulièrement difficiles à solubiliser, ce qui nécessite des techniques plus robustes telles que l&#39;utilisation de macération automatiséTing emballage étanche, tel que M TUBES ensemble avec un dissociateur Mac. En outre, des mesures de contrôle simples pour le stockage de la matière extraite et non extraite à -80 ° C peuvent faire la différence entre l&#39;obtention d&#39;étiquetage optimale immédiatement après l&#39;extraction et ayant pauvres résultats semaines plus tard. Méthodes de QFWB modernes se sont révélés être plus sensible pour capturer des différences subtiles dans l&#39;expression des protéines et permettent aux plus polyvalent simultanée double étiquetage 3 par rapport aux techniques plus anciennes comme ECL. Il est essentiel que les protocoles de transfert de Western sont robustes et facilement reproductible pour la quantification précise et l&#39;analyse statistique. Ce protocole est sensible et suffisamment robuste pour être utilisée en routine pour la détection de protéines à travers une série d&#39;échantillons de tissus et d&#39;espèces différentes et 3 permet la quantification des protéines de faible abondance et élevés à l&#39;intérieur de la même QFWB réduisant par conséquent l&#39;utilisation des consommables ainsi que le temps pour l&#39;expérience 1. 1 En outre, la sensibilité accrue de cette technique permet la validation de plus en plus populaire - études omiques 9,14 Cependant, l&#39;exactitude est cruciale et l&#39;inclusion de mesures de contrôle appropriées doivent être respectées évitant ainsi l&#39;acquisition de données erronées.

Western Blot (WB) est une technique d&#39;analyse développé à l&#39;origine dans les années 1970 pour déterminer la présence ou l&#39;absence d&#39;une protéine d&#39;intérêt dans un échantillon biologique complexe, comme un homogénat de tissu 1. Communément dénommé immunoblot des protéines, en raison de la clé interaction anticorps-antigène, la méthode consiste en cinq étapes distinctes: 1) la séparation électrophorétique des protéines par leur point isoélectrique; 2) le transfert à une nitrocellulose ou polyfluorure de vinylidène (PVDF); 3) le marquage en utilisant un anticorps primaire spécifique à la protéine d&#39;intérêt; 4) incubation avec un anticorps secondaire dirigé contre l&#39;anticorps primaire; et 5) la visualisation. méthodologie de visualisation a évolué avec le temps pour améliorer la sécurité et de la sensibilité. Certains des premiers Western Blots ont été réalisées en utilisant la radio balises marquées qui ensuite évolué vers les chemilluminescent plus largement utilisé (ECL) des méthodes colorimétriques et puis. Radioactivity a été marqué directement sur ​​les sondes pour des antigènes spécifiques alors que les méthodes colorimétriques et ECL utilisent une technique de marquage indirect avec un rapporteur enzymatique tel que la peroxydase de phosphatase alcaline ou de la streptavidine de raifort (HRP) 2. L&#39;intensité de l&#39;étiquetage du produit luminescent ou chromogène est mesurée en utilisant la densitométrie par lequel la force du signal, fort ou faible, plus ou moins indique la présence de la protéine d&#39;intérêt dans l&#39;échantillon. ECL est la méthode plus sensible et donc favorisée mais toutes les deux trois méthodes ont été initialement mis au point sur ​​le film à rayons X avec des techniques d&#39;imagerie numérique plus sophistiqué ensuite établies 3. L&#39;avancement de l&#39;imagerie numérique de WB a non seulement permis à un chercheur de déterminer la présence ou l&#39;absence de leur protéine d&#39;intérêt, mais a également permis une déduction au niveau de l&#39;expression d&#39;une protéine choisie lorsqu&#39;on les compare aux autres échantillons et peut donc être considéré comme «semi-quantitative &amp; #8221 ;. Récemment, une technique de transfert de Western véritablement quantitative et plus sensibles ont été mis au point de sorte que le niveau de fluorescence mesurée est directement liée à la quantité et à l&#39;expression d&#39;une protéine unique dans un échantillon: quantitatif fluorescent western blot (QFWB). Lorsque l&#39;on compare avec l&#39;étiquetage QFWB ECL, l&#39;utilisation d&#39;un anticorps secondaire fluorescent génère un profil de détection linéaire 4. Ceci est en contraste avec les techniques ECL où la linéarité du signal se produit généralement avec des charges faibles en protéines est inférieure à 5 ug et la saturation de signal directement liés à l&#39;expression des protéines, à savoir, avec des gènes domestiques exprimés de manière ubiquitaire 5,6. Cette disparité est très probablement dû à un plus grand nombre de sites de liaison disponibles pour un substrat ECL avidine à se lier à un secondaire biotinylé, ce qui entraîne un risque plus élevé de potentiel de saturation du signal. Ceci est l&#39;une des principales raisons de la base ECL immuno étant désignées comme étant ONLy &quot;semi-quantitative» 7. Le point de signal de saturation est d&#39;une importance critique pour la mesure des différences subtiles dans les niveaux d&#39;expression et peut conduire à des mesures inexactes. Au cours des dernières années, l&#39;avènement des techniques protéomiques répandues détaillant de plus en plus la sensibilité et l&#39;identification des différences d&#39;expression subtiles a entraîné une dépendance croissante sur vraiment quantitative Western blot pour des expériences de validation 8,9. L&#39;application de la méthodologie sensible, dynamique et véritablement quantitative est donc crucial. Beaucoup de &quot;optimisés&quot; méthodes de transfert de Western ont été utilisés par des laboratoires indépendants, qui se révèlent souvent difficiles à mettre en place ou de reproduire en raison d&#39;ajustements méthodologiques subtils qui peuvent ne pas être dans les protocoles officiels et documentés. Ceci est un protocole établi et robuste pour QFWB et fournit des stratégies utiles pour la résolution des problèmes communs tha plust peut survenir au cours de la mise en œuvre. Ce protocole a été optimisé pour une utilisation avec des homogénats de cerveau de souris, mais a depuis été utilisé efficacement dans un large éventail d&#39;échantillons de tissus et les espèces 4,9,10. Variations de protocole de potentiel nécessaires pour résoudre les problèmes spécifiques sont inclus.

<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

La fin des années 1970 ont vu la première utilisation déclarée publiquement du western blot, une technique pour évaluer la présence et l&#39;abondance relative des protéines spécifiques dans des échantillons biologiques complexes. Depuis lors, la méthodologie western blot est devenue un élément commun de la biologie moléculaire répertoire expérimental. Une recherche rapide de PubMed en utilisant le terme &quot;western blot&quot; suggère que plus de 220 000 manuscrits publiés ont fait usage de cette technique d&#39;ici l&#39;an 2014. Surtout, les dix dernières années ont vu des avancées d&#39;imagerie techniques couplées avec le développement marqueurs fluorescents de sensibles qui ont permis d&#39;améliorer la sensibilité et a donné encore plus de plages de détection linéaire. Le résultat est une fluorescence maintenant vraiment quantifiable fondé Western Blot (QFWB) qui permet aux biologistes d&#39;effectuer une analyse comparative de l&#39;expression avec une plus grande sensibilité et de précision que jamais auparavant. Beaucoup de &quot;optimisé&quot; western blotméthodes existent et sont utilisées dans différents laboratoires. Ceux-ci se révèlent souvent difficiles à mettre en œuvre en raison de l&#39;exigence de modifications procédurales subtiles mais sans papiers. Ce protocole fournit une description complète de la méthode d&#39;QFWB établi et robuste, avec des stratégies de résolution des problèmes.

Comme la sensibilité à QFWB et la plage linéaire de détection est supérieure à la détection ECL classique, il ya un certain nombre de mesures de contrôle qui sont essentiels pour assurer l&#39;exactitude des données sont collectées, facilitant ainsi l&#39;interprétation efficace. Tout d&#39;abord, l&#39;inclusion des échantillons témoins positifs comme représenté sur la figure 1. D&#39;autre part, l&#39;optimisation du transfert de garantir un mouvement équivalent de protéines de haut et de bas poids moléculaire à partir du gel à la membrane comme présenté dans la figure 2. En troisième lieu, l&#39;optimisation d&#39;anticorps, en particulier secondaire des anticorps dont l&#39;optimisation est souvent négligé, mais qui peuvent produire non spécifique bandes capables d&#39;interférer avec l&#39;interprétation correcte de la protéine (s) d&#39;intérêt. Voir Figure 3. Quatrièmement, il peut aussi être le cas que quand une protéine apparaît indétectable mais devrait être présente, cela peut aussi être une question d&#39;anticorps secondaire qui peut être corrigé en utilisant simplement un raïs secondairesed dans un hôte de différentes espèces. Voir figure 4. Cinquièmement, l&#39;étiquetage de la protéine totale et l&#39;analyse est une méthode beaucoup plus robuste et quantifiable par rapport à l&#39;utilisation unique de protéine traditionnel (s) qui sont exprimés de façon ubiquitaire à des étalons de référence internes 3. Beaucoup de ces protéines simples ont été trouvés à être exprimés de manière différentielle dans des modèles de maladies neurodégénératives, ainsi qu&#39;entre les échantillons de tissus différents et l&#39;uniformité d&#39;expression peut modifier dans le même tissu 3. Par conséquent, la production d&#39;un gel de commande de charge confirme l&#39;uniformité de la charge de l&#39;échantillon lorsqu&#39;il est combiné avec une analyse des protéines totales par la comparaison et la quantification de la charge de protéine dans chaque ligne à différentes gammes de poids moléculaires mesurés par rapport à chaque échantillon pour indiquer l&#39;erreur standard comme le montre la figure 6 . Fait important, l&#39;ensemble de ces techniques et des contrôles de dépannage ne sont efficaces que la sensibilité et la cohérence de l&#39;analyse tutils appliquées par l&#39;opérateur (figure 5). Enfin cette technique se prête bien à l&#39;arrachement et re-sondage de membranes avec plus de souplesse que ECL en raison de facteurs y compris, mais sans s&#39;y limiter, une sensibilité accrue, de fond réduit, la détection de double couleur et la stabilité de la membrane dans des conditions de stockage à long terme. Voir la Figure 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.

Un Guide to Modern quantitative fluorescente Western blot avec des stratégies de dépannage

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 ...

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50.9K vues.  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.

Vidéo: 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 blot en utilisant l&#39;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 ...

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Biologie

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 Blot: préparation d&#39;échantillons à la détection

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: Protocole Basic Plus Stratégies de dépannage et d&#39;optimisation

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.

Flux de travail sans taches V3 pour un contrôle pratique, pratique et fiable de la charge totale en protéines dans 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 ...

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;

Un guide pratique pour Phylogenetics pour non-experts

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.

Le plus rapide de l&#39;Ouest dans la ville: une touche contemporaine sur l&#39;analyse classique 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.

Floral-Dip Transformation de lin (<em&gt; Linum usitatissimum</em&gt;) Pour générer transgéniques descendances avec un taux de transformation élevé

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.

Code-barres génétique avec protéines fluorescentes pour des applications multiplexés

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.

Développement d&#39;un Quantitative recombinase polymérase L&#39;amplification Assay avec un contrôle positif interne

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

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

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

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.

Mucine Agarose Gel Electrophoresis: Western Blot pour les glycoprotéines de haut poids moléculaire,


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