Name: ultra-high-speed western blot using immunoreaction enhancing technology
Uploaded: 2020-09-26T13:00:00.000+00:00
Duration: 5 min 59 s
Description: Watch this Scientific Journal Video about Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology at JoVE.com

Bu çalışma Intramural Araştırma Bölümü, Ulusal Kalp, Akciğer ve Kan Enstitüsü, Ulusal Sağlık Enstitüleri tarafından desteklendi. S.H., Japonya Kamu-Özel Ortaklığı Öğrenci Yurtdışında Eğitim Programı tarafından desteklendi ve H.N. ve K.Y. Valor ve V Drug Overseas Training Scholarship tarafından desteklendi.

1. SDS-PAGE ve bir PVDF membran aktarın
<ol><li>Proteinleri göreli boyuta göre ayırmak için SDS-PAGE gerçekleştirin. NOT: Herhangi bir ticari veya ev yapımı jel sistemi iyidir. Lütfen üreticinin protokolüne uyun.</li>
	<li>Ayrılmış proteinleri bir jelden PVDF zarına aktarın. NOT: Yaygın transfer yöntemleri (yarı kuru veya ıslak transfer) uygundur. Lütfen üreticinin protokolüne uyun.</li>
</ol>2. PVDF membranlarının engellenmesi
<ol><li>Membranları oda sıcaklığında ajitasyon ile 1 saat boyunca uygun engelleme tamponlarında kuluçkaya yatırın. NOT: Birincil antikor ve algılama sistemine bağlı olarak uygun bir blokaj tamponu seçilmelidir. Örneğin, en tipik blokerler BSA, yağsız kuru süt, kazein ve ticari sentetik polimerlerdir. %0,1 Ara20 ile PBS ve/veya TBS en sık kullanılan arabelleklerdir. Bu engelleme adımı 4 °C'de bir gecede yapılabilir.</li>
</ol>3. Primer antikor ile inkübasyon
<ol><li>Damıtılmış su ile %10 immünoreaksiyon arttırıcı ajan-1 çözeltisi (IRE-1) hazırlayın. Girdap iyi.</li>
	<li>Seyreltilmiş primer antikoru% 10 IRE-1 ile hazırlayın. Hafifçe ve iyice karıştırın.</li>
	<li>Daha büyük bir membran (4 cm x 8 cm – 8 cm x 8 cm) kullanıyorsanız, 50 mL konik santrifüj tüplerine 8 mL antikor çözeltisi ekleyin. Daha küçük bir membran (2 cm x 8 cm – 4 cm x 8 cm) kullanıyorsanız, 14 mL yuvarlak tabanlı polipropilen tüpe(Malzeme Tablası)3 mL antikor çözeltisi ekleyin.</li>
	<li>PVDF membranını cımbızla alın ve engelleme solüsyonunu kısa bir süre boşaltın. PVDF zarını eldivenli parmaklarla bu tüpe yerleştirin ve tüm zarın tüpün duvarına yapıştığından emin olun. PVDF membran bir tüpe yerleştirildiğinde, başlangıçta jel ile içe doğru temas eden zarın "protein tarafı" ile yüzleşin. Kapağı sıkıca kapatın.</li>
	<li>50 mL'lik tüpü hibridizasyon fırını için bir cam şişeye yerleştirin. NOT: Bu inkübasyon için 14 mL tüp kullanıldığında, iç tüpü 50 mL tüpün ortasında tutmak için bir çift plastik halka tutucu kullanarak 50 mL'lik tüpe 14 mL'lik bir tüp yerleştirin.</li>
	<li>Hibridizasyon fırınını açın.</li>
	<li>Membranı en az 5 dakika boyunca 6 rpm dönüşle kuluçkaya yatırın. NOT: Membranın her zaman duvara yapıştığından ve (iç) tüpün membranı antikor çözeltisi ile eşit şekilde kaplayacak şekilde yatay olduğundan emin olun. Gerçek deneyden önce antikorun seyreltilmesini ve kuluçka süresini kalibre edin.</li>
</ol>4. Membran yıkama
<ol><li>Dönüşü durdurun.</li>
	<li>PVDF membranını cımbızla tüpten dikkatlice çıkarın ve membranı 50 mL PBS-T içeren bir kaba yerleştirin.</li>
	<li>Membranı PBS-T ile kısa bir süre durulayın.</li>
	<li>Zarı kabarmış su ile kabarcık görününe kadar durulayın. Bu antikorun çoğunluğunu çıkarmaktır.</li>
	<li>PVDF membranını kaptan 250 mL PBS-T içeren salata spinner'a aktarın.</li>
	<li>Süzgeç sepetini spinner'a yerleştirin. Kapağın güvenli bir şekilde takıldığından emin olun.</li>
	<li>Spinner'ı etkinleştirin ve yaklaşık 20-30 s boyunca çalıştırın.</li>
	<li>Çözeltiyi atın ve spinner'ın içini damıtılmış suyla kısa bir süre durulayın.</li>
	<li>Spinner'a tekrar 250 mL PBS-T ekleyin.</li>
	<li>4.6 ve 4.7 adımlarını yineleyin.</li>
</ol>5. İkincil antikor ile inkübasyon
<ol><li>Damıtılmış su ile %10 immünoreaksiyon arttırıcı ajan-2 çözeltisi (IRE-2) hazırlayın. Girdap iyi.</li>
	<li>Seyreltilmiş ikincil antikorun %10 IRE-2 ile hazırlanması. Hafifçe ve iyice karıştırın.</li>
	<li>Adım 3.3'te belirtildiği gibi antikor çözeltisinin ve tüpün hacmini seçin.</li>
	<li>PVDF zarını cımbızla alın ve çözeltiyi kısaca boşaltın. 3.4. adımda belirtildiği gibi PVDF membranını tüpe yerleştirin.</li>
	<li>50 mL'lik tüpü hibridizasyon fırını için bir cam şişeye yerleştirin.</li>
	<li>Hibridizasyon fırınını açın.</li>
	<li>Membranı en az 5 dakika boyunca 6 rpm dönüşle kuluçkaya yatırın. Zarın her zaman duvara yapıştığından ve (iç) tüpün membranı antikor çözeltisi ile eşit şekilde kaplayacak şekilde yatay olduğundan emin olun. NOT: Gerçek denemeden önceki kuluçka süresini kalibre edin. İkincil antikor floresan konjuge edildiğinde, inkübasyonu karanlıkta gerçekleştirin.</li>
</ol>6. Membran yıkama
<ol><li>4.1 ile 4.10 arasında olan adımları izleyin.</li>
</ol>7. Görüntü alımı
<ol><li>Chemiluminescent algılama<ol>
<li>Düz bir yüzeye bir parça yarı nakil esnek film (10 cm x 15 cm) yerleştirin.</li>
		<li>Chemiluminescent substratının 2 bileşenini 5 mL'lik bir tüpte karıştırın.</li>
		<li>Karışık substratın 1,5 mL'sini hemen yarı nakil esnek filme aktarın ve üzerine PVDF zarını yerleştirin. Daha sonra, substrat çözeltisinin geri kalanını zara aktarın.</li>
		<li>PVDF membranı, yarı saydam esnek bir film üzerindeki karışık substrat ile 1 dakika boyunca kuluçkaya yatırın.</li>
		<li>PVDF membranını cımbızla alın ve substrat çözeltisini kısa bir süre boşaltın.</li>
		<li>Membranı bir çift şeffaflık filmi arasına sıkıştırın.</li>
		<li>Görüntüyü chemiluminescent modu altında alın.</li>
	</ol>
</li>
	<li>Floresan algılama<ol>
<li>PVDF zarını cımbızla alın ve çözeltiyi kısaca boşaltın.</li>
		<li>Membranı bir çift şeffaflık filmi arasına sıkıştırın.</li>
		<li>Görüntüyü floresan modunda elde edin. NOT: Maksimum hassasiyet sağlamak için 7.1 ve 7.2 adımları görüntüleme makinesine yakın bir yerde yapılmalıdır.</li>
		<li>Arka plan sinyali yüksek olduğunda, 80-100 mL PBS-T içeren bir kapta yıkama adımı gerçekleştirin: birincil antikor için 3 kez 10 dakika durulayın ve ikincil antikor için 5 dakika durulayın. NOT: %10 IRE çözeltileri ile seyreltilmiş hem birincil hem de ikincil antikorlar hassasiyet kaybı olmadan 8-10 kata kadar kullanılabilir6. Çözelti 1 aya kadar 4 °C'de tutulmalıdır.</li>
	</ol>
</li>
</ol>

<ol>
	<li>MacPhee, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+considerations+for+improving+Western+blot+analysis.">Methodological considerations for improving Western blot analysis.</a> Journal of Pharmacol Toxicology Methods. 61 (2), 171-177 (2010).</li><li>Janes, K. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems+analysis+of+EGF+receptor+signaling+dynamics+with+microwestern+arrays.">Systems analysis of EGF receptor signaling dynamics with microwestern arrays.</a> Nature Methods. 7 (2), 148-155 (2010).</li><li>Treindl, F., 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=A+bead-based+western+for+high-throughput+cellular+signal+transduction+analyses.">A bead-based western for high-throughput cellular signal transduction analyses.</a> Nature Communications. 7, 12852 (2016).</li><li>Sajjad, S., Do, M. T., Shin, H. S., Yoon, T. S., Kang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+western+blot+assay+by+rotational+cyclic+draining+and+replenishing+procedure.">Rapid and efficient western blot assay by rotational cyclic draining and replenishing procedure.</a> Electrophoresis. 39 (23), 2974-2978 (2018).</li><li>Kurien, B. T., Scofield, R. 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Yazarlar, bu makalenin içeriğiyle doğrudan ilgili rakip çıkarlar beyan etmemektedir.

Batı blotu, ~40 yıl önce7,8. O zamandan beri, teknik gelişmeye devam etti, sonraki yenilikler tekniğin hassasiyetini, hızını ve niceliğini geliştirdi2, 9,10,11,12,13. Burada sunulan gelişmiş ultra yüksek hızlı batı şişkinlik protokolü, mevcut teknik...

Batı lekesi (immünoblot olarak da bilinir), çok çeşitli bilimsel ve klinik disiplinlerde güçlü ve temel bir tekniktir. Bu teknik, proteinlerin varlığını, bağıl bolluğunu, bağıl moleküler kütlesini ve çeviri sonrası modifikasyonlarını incelemek için kullanılır1. Dijital görüntü analizi ile birlikte, bu yöntem proteinlerin bolluğunu ve protein modifikasyonlarını güvenilir bir şekilde analiz edebilir2. Batı lekesi rutin olarak yapılsa da zaman alan ve emek yoğun bir yöntemdir. Antikorun membranlarla uzun inkübasyonları gereklidir. Burada, duyarlılıktan ödün vermeden bu sınırlamanın üstesinden gelen kuluçka yönteminin bir değişikliğini açıklıyoruz.
Membran inkübasyonu sırasında, antikorlar çözelti içinde yüzerken, antijenler membran üzerinde hareketsiz hale getirilir. Yüksek benzeşimleri nedeniyle, antijene antikor bağlanma hızı, antikorların dökme çözeltiden zara yayılmasından daha hızlıdır. Bu, düşük konsantrasyonda bir "tükenme katmanı" oluşturur (Şekil 1). Bu teknikte uzun kuluçka sürelerinin ana faktörü olan pasif difüzyon yoluyla daha uzak antikorların zara ulaşması saatler alabilir. Bu etkiye toplu taşıma sınırlaması (MTL)3denir. Antikor içeren çözeltinin tekrar tekrar boşaltılması ve yenilenmesinin tükenme tabakasını bozabileceği ve MTL4'ünüstesinden gelebileceği önerilmiştir. Burada, geleneksel bir batı blotting protokolünde MTL'nin etkisini azaltmak ve gerekli kuluçka süresini kısaltmak için bu döngüsel drenaj ve ikmal (CDR) kavramını uygulayan benzersiz bir teknik tasarladık.
Algılama hassasiyetini kısa bir kuluçka süresiyle korumak için, antijen-antikor reaksiyonunu hızlandıran ve böylece sinyal-gürültü oranını artıran bir immünoreaksiyon arttırıcı teknoloji kullandık5. Piyasada bulunan birçok immünoreaksiyon arttırıcı madde (IRE) 2 bileşenden oluşur (Çözüm 1 ve 2, bkz. Malzeme Tablosu). IRE'nin tescilli bileşimi veya etki mekanizması açıklanmadı, ancak daha önce IRE'nin katı faz bağlama testlerinde antijen ve antikor arasındaki ayrışma sabitini azalttığını bulduk, bu da artan benzeşimin en azından kısmen IRE6'nınarttırıcı etkisinden sorumlu olduğunu gösteriyor. CDR'nin IRE ile kombinasyonu, hassasiyettan ödün vermeden tüm prosedür süresini azaltan ultra yüksek hızlı bir batı blotting protokolü verir.

<table><tbody><tr><td>14 mL Round Bottom High Clarity PP Test Tube, Graduated, with Snap Cap</td><td>Falcon</td><td>352059</td><td></td></tr><tr><td>Apollo Transparency Film for Laser Printers</td><td>N/A</td><td>N/A</td><td>Any kinds of Laser Printer Transparency Film are fine.</td></tr><tr><td>Azure Imaging System 600</td><td>Azure Biosystems</td><td>Azure 600</td><td>Any kind of Image Acquiring Sytem is fine for chemiluminescent and/or fluorescent detection.</td></tr><tr><td>Can Get Signal Immunoreaction Enhancer Solution</td><td>TOYOBO</td><td>NKB-101</td><td></td></tr><tr><td>CARNATION Instant Nonfat Dry Milk</td><td>Carnation</td><td>N/A</td><td></td></tr><tr><td>ECL anti-mouse IgG, Horseradish peroxidase linked F(ab&amp;rsquo;)2 fragment from sheep</td><td>GE Healthcare</td><td>NA9310V</td><td></td></tr><tr><td>ECL anti-rabbit IgG, Horseradish peroxidase linked F(ab&amp;rsquo;)2 fragment from donkey</td><td>GE Healthcare</td><td>NA9340V</td><td></td></tr><tr><td>HYBAID Micro-4</td><td>N/A</td><td>N/A</td><td>Any hybridization oven is fine as long as the tubes are rotated evenly at a horizontal position.</td></tr><tr><td>Immobilon-P PVDF Membrane, 0.45 &amp;micro;m pore size</td><td>Millipore Sigma</td><td>IPVH304F0</td><td></td></tr><tr><td>IRDye 680RD Goat anti-Mouse IgG Secondary Antibody</td><td>LICOR</td><td>926-68070</td><td></td></tr><tr><td>IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody</td><td>LICOR</td><td>926-32211</td><td></td></tr><tr><td>KPL LumiGLO Reserve Chemiluminescent Substrate</td><td>seracare</td><td>5430-0049</td><td></td></tr><tr><td>Mouse anti-&amp;szlig; actin antibody</td><td>Millipore Sigma</td><td>A5316</td><td></td></tr><tr><td>Odyssey Blocking Buffer (PBS)</td><td>LICOR</td><td>927-40100</td><td>Blocking Buffer for fluorescent detection</td></tr><tr><td>OXO Salad Spinner</td><td>OXO</td><td>32480V2B</td><td>Any salad spinner is fine as long as the PVDF membranes are rinsed vigorously without tear.</td></tr><tr><td>Parafilm Sealing Film</td><td>Bemis</td><td>Parafilm M PM996</td><td>semi-transparent flexible film</td></tr><tr><td>Polyethylene Flat-Top Screw Caps for 50 mL Conical Bottom Centrifuge Tubes</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Rings to hold 14 ml tube in the center of 50 ml tube</td><td>N/A</td><td>N/A</td><td>Prepared in a machine shop</td></tr><tr><td>Rabbit anti-6X His tag antibody</td><td>Abcam</td><td>ab9108</td><td></td></tr><tr><td>Tween20</td><td>Millipore Sigma</td><td>P2287</td><td></td></tr></tbody></table>

ultra-high-speed western blot using immunoreaction enhancing technology

Batı lekesi (immünoblot olarak da bilinir) biyomedikal araştırmalar için kurallı bir yöntemdir. Genellikle belirli proteinlerin bağıl boyutunu ve bolluğunu ve çeviri sonrası protein modifikasyonlarını belirlemek için kullanılır. Bu teknik zengin bir geçmişe sahiptir ve sadeliği nedeniyle yaygın kullanımda kalır. Bununla birlikte, batı şişkinlik prosedürünün tamamlanması saatler, hatta günler alır ve kritik bir darboğaz, verimini sınırlayan uzun kuluçka süreleridir. Bu inkübasyon adımları, antikorların toplu çözeltiden membran üzerindeki hareketsiz antijenlere kadar yavaşça yayılması nedeniyle gereklidir: zarın yakınındaki antikor konsantrasyonu, toplu konsantrasyondan çok daha düşüktür. Burada, antikor çözeltisinin döngüsel drenajı ve yenilenmesi (CDR) yoluyla antijen bağlanmasını iyileştirerek bu kuluçka aralıklarını önemli ölçüde azaltan bir yenilik sunuyoruz. Ayrıca tahlil hassasiyetini korumak için bir immünoreaksiyon arttırıcı teknoloji kullandık. CDR yönteminin ticari bir immünoreaksiyon arttırıcı madde ile kombinasyonu çıkış sinyalini artırdı ve antikor inkübasyon süresini önemli ölçüde azalttı. Elde edilen ultra yüksek hızlı batı lekesi, hassasiyet kaybı olmadan 20 dakikada gerçekleştirilebilir. Bu yöntem hem chemiluminescent hem de floresan tespiti kullanılarak batı lekelerine uygulanabilir. Bu basit protokol, araştırmacıların birçok örnekte protein ekspresyonunun analizini daha iyi keşfetmelerini sağlar.

Bu örnek, CDR yönteminin batı blot üzerindeki immünoenhancing teknolojisi ile birlikte etkinliğini göstermektedir. Statik inkübasyon, PVDF membranlarının antikor çözeltisi ile inkübe edildiği yarı saydam esnek bir film üzerinde yapılırken, CDR inkübasyonu membranları ve antikor çözeltisini (Film) içeren tüpleri döndürerek hibridizasyon fırınında yapıldı. Şekil 2'de batı lekelerinde chemiluminescent tespiti için gerekli olan antijen ve antikor miktarları gö...

Watch this Scientific Journal Video about Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology at JoVE.com

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

Ultra yüksek hızlı batı şişkinlik tekniği, bir immünoreaksiyon arttırıcı madde ile birlikte döngüsel drenaj ve ikmal (CDR) teknolojisi ile antijen-antikor bağlama kinetiğinin iyileştirilmesi ile geliştirilmiştir.

İmmünreaksiyon Arttırıcı Teknoloji Kullanan Ultra Yüksek Hızlı Batı Blot

A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and ...

ultra-high-speed-western-blot-using-immunoreaction-enhancing

Research

JoVE Journal

Biochemistry

5.8K Görüntüleme.  National Institutes of Health. Ultra yüksek hızlı batı şişkinlik tekniği, bir immünoreaksiyon arttırıcı madde ile birlikte döngüsel drenaj ve ikmal (CDR) teknolojisi ile antijen-antikor bağlama kinetiğinin iyileştirilmesi ile geliştirilmiştir.

Video: Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

Kızılötesi Floresan Boya-etiketli oligonükleotidler kullanılarak Elektroforetik Hareketlilik Kaydırma Testi için optimize Protokol

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

Biyokimya

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

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

Proteinler ve onların izoformlarının kapiller Isoelectric odaklanarak yöntemi tarafından son derece hassas ve nicel tespiti

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides ...

Immunoblot Analysis

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. It involves the solubilization and electrophoretic separation of proteins, glycoproteins, or lipopolysaccharides by gel electrophoresis, followed by quantitative transfer and irreversible binding to nitrocellulose, PVDF, or nylon. The immunoblotting technique has been useful in identifying specific antigens recognized by polyclonal or monoclonal antibodies and is highly sensitive (1 ng of antigen can be detected). This unit provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. This video provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

Immüno Analizi

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the ...

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 ...

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 ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

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 within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete 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 ...

Immunoprecipitation-Based Techniques: Purification of Endogenous Proteins Using Agarose Beads

Source: Susannah C. Shissler1, Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Immunoprecipitation (IP, also known as a &#39;pull-down&#39; assay) is a widely used technique that has applications in a variety of fields. First conceived in 1984, it was refined in 1988 (1, 2). The fundamental goal of IP is purification and isolation of a specific protein using an antibody against that protein. The word &#34;immuno&#34; refers to the use of an antibody while the word &#34;precipitation&#34; refers to pulling down a specific substance from a solution. The target protein might be endogenous or recombinant. Most recombinant proteins have an epitope tag (i.e. myc or flag) attached to them to simplify subsequent purification. Typically, it is easier to optimize recombinant protein IP because the antibodies against recombinant epitope tags are very strong and effective. Antibodies against endogenous proteins have extremely variable efficacy - making it much more difficult to optimize these IPs. A necessary step after immunoprecipitation is verification of purification. The isolated protein is resolved using SDS-PAGE and subsequently probed for purity by western blots (Figure 1). An important control is the use of a different antibody during the Western blot to verify pull down of the correct protein. The combination of IP with subsequent techniques is a powerful analysis tool. The goal after purification may be characterization of the protein itself by NMR, mass spectrometry, and in vitro assays, or analysis of the protein&#39;s interacting partners (i.e. protein, DNA, RNA) (3, 4, 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10502/10502fig1.jpg" /> 
Figure 1: Overview of Immunoprecipitation Procedure. Immunoprecipitation is the isolation of a specific protein using an antibody. After production of lysate from cells, there are two major steps- pre-clearing and pull down. During pre-clearing step, the cell-lysates are pre-cleared of proteins that bind to antibodies non-specifically using an isotype control antibody. In pull down step, the target protein is pulled down using a protein-specific antibody. The isolated protein is then analyzed by Western blot. Isotype antibodies and protein specific antibodies have the same constant domain, but different antigen binding domains. A key component of this protocol is Protein A/G agarose beads that bind the constant domain of antibodies- allowing immunoprecipitation of the target protein. <a href="https://www.jove.com/files/ftp_upload/10502/10502fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Antibodies are the key component of an immunoprecipitation that differentiate it from other forms of protein purification (i.e. nickel affinity column purification). Antibodies are molecules made by B cells that can recognize specific protein epitopes. Antibodies have two domains: constant (Fc) and antigen binding (Fab) (Figure 1). The constant domain identifies the type of antibody and dictates function in vivo. Usually, the constant domains of antibodies used for IP are mouse, rat, or rabbit IgG. The antigen binding portion of the antibody recognizes a specific epitope of a specific protein. Antibodies can recognize epitopes on folded proteins that may not exist when the protein is denatured and vice versa. Therefore, the availability of the epitope depends on protein folding - identifying an important factor to consider when choosing antibodies and conditions for IP.
Both prokaryotic and eukaryotic systems have antibody-binding proteins. In eukaryotic systems, the purpose is immune protection from bacteria while in prokaryotic systems, the purpose is protection from the immune system. Antibody-binding proteins affect IP methodology in two ways. First, there is a necessary pre-clearing step (Figure 1) to rid the lysate of proteins that bind antibodies - thereby reducing non-specific binding in the final product. This step uses an isotype antibody that has the same constant domain as but a different antibody binding domain than your protein-specific antibody. Bacterial antibody-binding proteins are the second key component of this method. After the protein-specific antibody binds the target protein, the antibody: protein complex must be pulled down (Figure 1). Proteins A, G, and L are bacterial proteins that bind the constant domain of antibodies. While bacteria use this to subvert the immune system, researchers have co-opted this system for easy antibody purification, and it is used during both the pre-clearing and pull-down steps. These proteins have different binding affinities for different species and different constant domain subtypes - another factor to consider when choosing conditions for IP. Many companies sell Protein A/G labeled agarose beads (Figure 1), pre-made spin columns, or resins to make columns. In general, beads and spin columns are used for smaller sample sizes while resins are used for bulk purification.
In this lab exercise, we demonstrate how to purify the endogenous protein c-myc, from primary murine thymocytes, using Protein A/G Plus agarose beads based basic immunoprecipitation technique. The protocol starts from cell lysate preparation and ends with the with verification of successful protein pull down using Western blot analysis.

Immunoprecipitation Techniques: Purification of Proteins

Source: Susannah C. Shissler1, Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Immunoprecipitation ...

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The Western Blot

Western Blotting is used to identify the presence of specific proteins in electrophoretically separated samples. Following separation by a technique known as sodium dodecyl sulfate polyacrylamide gel electrophoresis, or SDS-PAGE, western transfer is used to move proteins from a polyacrylamide gel onto a piece of membrane which traps the proteins in their respective locations. Next, the membranes are probed with antibodies in a process called immunboblotting. Immunoblotting uses antibody-protein and antibody-antibody binding through specific recognition sites, providing the high specificity required for identifying a single protein. The detection of antibodies takes place using reporter systems which includes the use of enzymes. Enzymes can be attached to the end of an antibody and react with substrates to produce changes in color or light. These signals can then be imaged and quantified using a process called densitometry.
This video-article presents an overview of the western blot technique by describing western transfer, the use of antibody detection, and image analysis. The steps involved with western transfer such as the assembly of the transfer sandwich and transfer conditions are discussed in detail as well as the theory behind antibody binding and detection of those antibodies. The broad applications of this technique are described through several examples including the detection of protein-protein interactions and identification of individual proteins within protein complexes.

Western Blotting: Western Transfer, Antibody Detection, and Image Analysis

Western Blotting is used to identify the presence of specific proteins in electrophoretically separated samples.  Following separation by a technique ...

Temel Biyoloji

Hücresel ve Moleküler Biyolojide Temel Yöntemler

Chemiluminescent Western Blot Assay for Protein Processing

Source: Hillert-Richter, L. K. et al., <a href="https://app.jove.com/v/62842">Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex.</a> J. Vis. Exp. (2021).
This video describes the chemiluminescence-based western blotting of immunoprecipitated lysates. The technique helps determine protein processing and activation. The chemiluminescent signal detected is proportional to the level of protein processing during its activation.

Source: Hillert-Richter, L. K. et al., Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex. J. ...