Biz sayesinde mali destek için aşağıdaki istiyorum: BBSRC Enstitüsü Stratejik Program Harçlar - CF &amp; TMW; BBSRC Doğu Bio DTP finansman - LG; Edinburgh Darwin Güven - MLH. Biz de bu metinde TREM2 optimizasyonu eklemek için izin Dr. Barry MCCOLL teşekkür etmek istiyorum.

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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.

Nedeniyle göz ve planlama, herhangi bir deney öncesinde esastır ve sonuçta kullanılan tekniğin başarısını belirlemek olabilir. Uygun antikorlar, transfer ve görüntüleme yöntemleri kullanmayı tercih çalışırken potansiyel engellerden bir bolluk sunabilir WB kullanarak protein algılama gelişmeler. Neyse ki, protein varlığı ve örnekler arasındaki giderek ince anlatım farklılıkları belirlemek için rutin olarak kullanılabileceği QFWB dikkatli bir kontrol listesi ve uygun kontrol önlemleri kullanarak. Bu protokol önlemek ve / veya onunla ilişkili birçok ortak tuzaklar bazılarının üstesinden gelmek için floresan nicel batı lekeleme yanı sıra birkaç sorun giderme stratejileri kapsamlı bir kılavuz sağlar. Duyarlılığı korumak ve elde etmek için kullanılan kritik adımlar gerçekten ölçülebilir ve karşılaştırılabilir ölçümler şunlardır: doku örneklerinden 1) sağlam protein izolasyonu; 2) numune hazırlama; 3) doğru bir proteini loading toplam protein analizi ile belirlendi; Ben-Blot kullanarak proteinlerin 4) Optimal transfer; Bir termal görüşleri ve ilgili yazılım kullanılarak,% 0.1 Tween20 içeren bloke edici tampon içinde birincil ve ikincil antikorlar 5) hazırlanması, ve 6) doğru görselleştirme ve analiz. Kızılötesi floresan algılama gerçekten kantitatif ve daha geleneksel ECL algılama tekniklerle 3,11 ile karşılaştırıldığında daha fazla hassasiyet sağlar. Bu algılama sistemi çok yüzlü olan ve bu nedenle QFWB sınırlı değildir. Bu sistem, tam doku bölümleri 12 görselleştirme ve sayılmasına olanak tanıyan düşük güçte immünohistolojik etiketleme görüntüleme yeteneğine sahiptir. Bu nicel değerlendirme açısından geleneksel mikroskopları ile geleneksel immunofloresan yakalayan rakip kadar kırmızı görüntüleme görebiliyordu çözünürlük açısından gelecekteki potansiyel gelişim bir alandır. Bununla birlikte, p içerisindeki hafif değişiklikler, daha büyük hassasiyet ileminimum ve kontrol önlemlerine tutulur ifade bu çeşitliliği sağlamak açısından önemli olduğunu rotein sağlam protokolleri ile sıkıdır. Bu örnek yükleme düzgün, algılama gerçek ve ilgili üreticinin kullanım kılavuzu test aktarmak için olup olmadığını belirlemek için, birincil ve ikincil antikorlar optimizasyonu olduğu güvence sağlamak için toplam protein lekeli jellerin üretimi ve ardından bu doku örneğinin titiz protein ekstre etme ile başlar Zaman verimli protein transferini elde etmek. Yine de, WB koşulları optimize edilmiş bile, hala sorunlar tam burada keşfedilmiş olmayabilir western&#39;lerine çalışan ile orada ilişkili olabilir. Bunlara aşağıdakiler dahildir fakat Protein çözünmüş ve ekstraksiyon tamponu seçimi dahil çeşitli faktörlere bunlarla sınırlı değildir. Bazı tampon protein konsantrasyonu deneyleri engelleyebilir ve bazı dokular gibi otomatik MACERA kullanılması gibi daha sağlam teknikler gerektiren, çözünür hale getirmek için özellikle zordurting böyle bir Mac&#39;ler dissociator ile birlikte M tüpler gibi kapları kapalı. Buna ek olarak, -80 ° C&#39;de ayıklanır ve non-çıkartılan malzemenin depolanması için basit kontrol tedbirleri hemen çekimi sonrası optimum etiketleme elde edilmesi ve kötü sonuç hafta sonra olması arasında fark olabilir. Modern QFWB yöntemleri protein ifadesindeki ince farklılıkları yakalamak için daha duyarlı olduğu kanıtlanmış ve bu ECL gibi eski tekniklere göre daha çok yönlü eşzamanlı ikili etiketleme 3 izin veriyoruz. Bu batı lekeleme protokolleri sağlam ve doğru ölçümü ve istatistiksel analiz için kolayca tekrarlanabilir olmaları önemlidir. Bu protokol, farklı doku örneklerinin ve türler 3 çeşitli boyunca proteinlerinin tespiti için rutin olarak kullanılacak yeterince hassas ve sağlamdır ve bu nedenle deney 1 &#39;deki sarf malzemesi kullanımını hem de süresini azaltmak, aynı QFWB içinde düşük ve yüksek bolluk proteinlerin ölçülmesine olanak sağlar. 9,14 Ancak hassasiyeti bu şekilde hatalı veri toplama kaçınarak uyulması gereken önemli ve uygun kontrol önlemlerinin dahil edilmesi omic çalışmaları - 1 Buna ek olarak, bu tekniğin artan duyarlılık giderek popüler doğrulama sağlar.

Western blot analizi (DB), orijinal olarak bir doku homojenatı 1 gibi, karmaşık bir biyolojik numunede ilgi konusu bir proteinin varlığını veya yokluğunu belirlemek için, 1970&#39;lerin geliştirilen bir analiz tekniğidir. Yaygın nedeniyle önemli antikor-antijen etkileşimi, protein immunoblot olarak anılacaktır, bu yöntemin 5 ayrı adımlardan oluşur: izoelektrik alanına göre proteinlerin 1) elektroforez yolu ile ayrılması; 2) nitroselüloz veya Polivinil difluoride (PVDF) membran transfer; 3), ilgi konusu proteine ​​özel bir birincil antikor ile etiketlenmesi; Birincil antikora karşı yönlendirilmiş bir ikincil antikorla 4) inkübasyon; ve 5) görselleştirme. Görselleştirme metodoloji güvenliği ve duyarlılığı artırmak için zamanla gelişti. İlk Kitaplarına bazıları, daha sonra daha yaygın olarak kullanılan chemilluminescent (ECL) metodları, kolorimetrik ve ilerlemiş radyo-etiketli etiketler kullanılarak gerçekleştirildi. Radioactivikolorimetrik ve ECL metodolojiler örneğin alkalin fosfataz ya da streptavidin-yaban turpu peroksidaz (HRP) 2 gibi bir enzim haberci ile dolaylı bir etiketleme tekniğini kullanırlar Ty doğrudan belirli bir antijen için problar üzerine etiketlendi. Chromagen ya da lüminesan ürünün etiketlenmesi yoğunluğu güçlü ya da zayıf bir sinyal gücü, örnek olarak, ilgi konusu proteinin daha fazla veya daha az varlığını belirtir, burada densitometrisi kullanılarak ölçülür. ECL daha hassas ve bu nedenle tercih edilen yöntem 2, ancak 3 yöntemleri ilk olarak daha sonra 3 Kurulan daha gelişmiş bir dijital görüntüleme teknikleri ile X-ışını filmi üzerinde geliştirilmiştir. WB dijital görüntüleme ilerlemesi sadece kendi ilgi proteinin varlığını veya yokluğunu belirlemek için, bir araştırmacı izin verilir, ancak başka numuneler karşı karşılaştırıldığında, aynı zamanda seçilmiş bir protein ekspresyon düzeyi için çıkarsama bırakıldı ve bu yüzden şu şekilde de ifade edilebilir &quot;Yarı-kantitatif &amp; #8221 ;. Ölçülen floresan seviyesinin doğrudan numune içindeki miktarı ve tek bir proteinin ekspresyonu ile ilgilidir, burada en son, gerçek bir nicel ve daha hassas Batı lekeleme tekniği geliştirilmiştir: nicel (QFWB) batı beneklemesinden floresan değildir. ECL etiketi ile QFWB karşılaştırıldığı zaman, bir fluorescent sekonder antikor kullanımı doğrusal tespit profili 4 oluşturur. Bu sinyal doğrusallık, genellikle her yerde ifade hazırlık geninin 5,6 ile örneğin, doğrudan protein ifadesi ile ilgili olan 5 ug ve sinyal doygunluk altında düşük protein yükleri ile oluşur ECL tekniklerine tersidir. Bu dengesizlik, muhtemelen potansiyel bir sinyal doyma daha yüksek bir olasılıkla edilen biyotinlenmiş sekonder bağlamak için bir avidin ECL alt-tabaka için müsait bağlanma mevkilerinin daha fazla sayıda neden olur. Bu gibi onl bahsedilerek ECL baz immünolekeleme için ana nedenlerinden biridiry &quot;yarı-kantitatif&quot; 7. Ekspresyon düzeylerinde ince farkları ölçmek ve doğru ölçümlerine neden zaman sinyalinin doyma noktası önem taşımaktadır. Son yıllarda giderek artan duyarlılık ve ince ifade farklılıklarının belirlenmesini detaylandırma yaygın proteomik tekniklerin gelişiyle doğrulama deneyleri 8,9 için gerçekten kantitatif batı lekeleme üzerinde gittikçe artan bir güven sonuçlandı. , Hassas, sağlam ve gerçekten kantitatif metodoloji uygulanması çok önemlidir. Pek çok &quot;optimize&quot; batı lekeleme metodolojileri nedeniyle sık sık resmi belgelenmiş protokollerde belirgin olmayabilir ince metodolojik ayarlamalar kurmak veya taklit edilmesi zor kanıtlamak bağımsız laboratuarlar tarafından kullanılmıştır. Bu QFWB için kurulan ve sağlam bir protokoldür ve ayrıca ortak sorunları tha gidermek için değerli stratejiler sunart uygulanması sırasında ortaya çıkabilir. Bu protokol başlangıçta murin beyin homojenatlarının ile kullanım için optimize edilmiş, ancak bu yana doku örnekleri ve türlerin 4,9,10 geniş bir dizi etkili kullanılmıştır. Belirli sorunların giderilmesi için gerekli potansiyel protokol varyasyonları dahildir.

<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

1970&#39;lerin western blot, varlığını ve karmaşık biyolojik örneklerin içindeki belirli proteinlerin göreceli bolluk değerlendirmek için bir tekniğin ilk halka bildirdi kullanımı gördüm. O zamandan beri, batı lekeleme metodoloji moleküler biyologlar deneysel repertuarı ortak bir bileşeni haline gelmiştir. Terimini &quot;western blot&quot; kullanırken PubMed&#39;e üstünkörü bir arama 220.000 yayınlanan yazılarda aşan önemlisi 2014 yılına kadar bu tekniğin kullanımını yapmış önerir, son on yıl kalkınma ile birleştiğinde teknik görüntüleme gelişmeler gördük daha iyi bir duyarlılıktır ve doğrusal algılama daha büyük aralıkları vermiştir duyarlı floresan etiketler. Sonuç biyologlar her zamankinden daha fazla hassasiyet ve doğruluk ile karşılaştırmalı ifade analizi yürütmek için izin verir Western Blot (QFWB) tabanlı bir şimdi gerçekten Ölçülebilir Floresans. Pek çok &quot;optimize&quot; batı lekelememetodolojileri vardır ve farklı laboratuarlarda kullanılmaktadır. Bunlar genellikle nedeniyle ince ama belgesiz usul değişikliklerinin ihtiyacına uygulamak zordur kanıtlamak. Bu protokol giderme stratejileri ile tam bir kurulan ve sağlam QFWB yönteminin açıklayıcı bir tarifini sağlar.

QFWB duyarlılık ve algılama doğrusal aralığı geleneksel ECL algılama daha büyük olduğu, dolayısıyla etkili yorumuyla yardım doğru veri toplanması sağlamak için önemli olan kontrol tedbirleri, bir dizi vardır. İlk olarak, pozitif kontrol numunelerinin dahil Şekil 1 &#39;de gösterildiği gibi. İkinci olarak, transfer optimizasyonu membrana jelden yüksek ve düşük moleküler ağırlıklı proteinlerin denk hareketini garanti altına almak için, Şekil 2&#39;de sergilenen. Üçüncü olarak, antikor optimizasyonu, özellikle orta kimin optimizasyonu genellikle gözden kaçan, ancak antikorlar ilgi protein (ler) doğru yorumlanması ile müdahale edebilen bantlama non-spesifik üretebilir. Şekil 3&#39;e bakın. Dördüncüsü, o da olabilir davayı bir protein saptanamayan görünür ama mevcut olması bekleniyor zaman, bu da sadece ikincil Rais kullanılarak düzeltilebilir ikincil antikor sorunu olabilir kifarklı bir tür konak ed. Şekil 4&#39;e bakınız. Beşinci olarak, toplam protein etiketleme ve analizi her yerde bulunan bir dahili referans standartları 3 olarak ifade edilmiştir geleneksel tek protein (ler) in kullanımı ile karşılaştırıldığında, çok daha sağlam ve ölçülebilir bir yöntemdir. Bu tür tek proteinlerin bir çok farklı şekilde, nörodejeneratif hastalıkların model yanı sıra arasında farklı doku örneklerinin ifade edildiği tespit edilmiştir ve ifade homojenliği, aynı doku 3 olan değiştirilebilir. Standart hata göstermek için karşılaştıran ve her numune karşı ölçülen, çeşitli molekül ağırlıkları aralıkları, her bir şeritte protein yükü miktarının bir toplam protein analizi ile birlikte, Şekil 6&#39;da gösterildiği gibi, Bu nedenle, yükleme kontrol jel üretim örneği yükün homojen teyit edecektir . Önemlisi, bu sorun giderme teknikleri ve tüm denetimleri sadece analiz t duyarlılığı ve tutarlılığı kadar etkilidiroperatör (Şekil 5) tarafından uygulanan ools. Son olarak, bu teknik, soyma ve bağlı faktörler de dahil olmak üzere, ancak, uzun süreli depolama koşulları altında artan bir hassasiyet, azaltılmış arka plan, iki renkli algılama ve zar stabilitesi bunlarla sınırlı olmamak üzere, ECL daha fazla esneklik membran yeniden sondalama için çok uygundur. Şekil 7 bakınız.

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.

Sorun Giderme Stratejileri Modern Kantitatif Floresan Western Blot A Guide

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 Görüntüleme.  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.

Batı Invitrogen NuPage Novex Bis Tris MiniGels kullanarak Blot

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

Biyoloji

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: Numune Hazırlama Algılama

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.

Polimeraz Zincir Reaksiyonu: Temel Protokol Plus, Sorun ve Optimizasyon Stratejileri

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.

Batı Blotting'de Pratik, Kullanışlı ve Güvenilir Toplam Protein Yükleme Kontrolü için V3 Lekesiz İş Akışı

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;

Nonexperts için Phylogenetics İçin Pratik Kılavuz

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.

Town Batı Hızlı: Klasik Western Blot Analizi Üzerine Bir Çağdaş Büküm

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.

Keten Çiçekli-Dip Dönüşüm (<em&gt; Linum usitatissimum</em&gt;) Yüksek Dönüşüm Oranı ile Transgenik progeny&#39;lerinde oluşturmak için

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.

Multiplexed Uygulamaları için Floresan Proteinleri ile Genetik Barkodlama

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.

İç Pozitif Kontrol ile Kantitatif Recombinase Polimeraz Amplifikasyon Testi Geliştirme

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.

Musin Agaroz jel elektroforezi: Yüksek molekül ağırlıklı glikoproteinler için Western Blot


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