Name: antibody labeling with fluorescent dyes using magnetic protein a and protein g beads
Uploaded: 2016-09-15T14:00:00.000+00:00
Duration: 6 min 48 s
Description: Watch this Scientific Journal Video about Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads at JoVE.com

Yüksek Kapasiteli Manyetik Protein A veya Manyetik Protein G Boncuk üzerine 1. Antikor Yakalama <ol><li> Düzgün nazik sallayarak manyetik boncuk yeniden askıya. boncuk aliquoting Süspansiyon üniforma tutun. </li><li> 1.5 ml mikrosantrifüj tüpüne boncuk çamuru 50 ul ekle. 10 saniye boyunca manyetik stand koyun. Dikkatle depolama tamponu çıkarın. </li><li> Antikor bağlama tamponu, 250 ul ekleyin. </li><li> Mix ve 10 saniye manyetik stand koyun. Dikkatle bağlayıcı tampon kaldırmak. </li><li> tanelerine antikorun 50-100 ug ihtiva eden numune 1.0 ml ilave edilir. Numuneler, hücre ortamında antikorlar ya da antikor arıtılabilir. </li><li> vorteksli bir mikserde veya son aşırı uç mikser kullanılarak, oda sıcaklığında 60 dakika boyunca örnek karıştırın. </li><li> 10 saniye boyunca manyetik stand tüp yerleştirin ve süpernatant kaldırmak. </li><li> antikor bağlama / yıkama tamponu 250 ul ilave et ve karıştır. 10 saniye boyunca manyetik stand koyun ve bağlayıcı / yıkama tamponu çıkarın. Bu s tekrarlayınİki yıkar toplam TEP. </li><li> amin kimyası veya Bölüm 3 tiol kimyası kullanılarak etiketli antikorlar kullanılarak etiket antikorlara Bölüm 2&#39;ye geçin. </li></ol> Amin kimyası kullanılarak 2. Antikor etiketleme <ol><li> Konjuge Antikor <ol><li> Boncuklar amin konjugasyon tampon 100 ul ekle. </li><li> boyanın 1.0 mg dimetil sülfoksit (DMSO), 100 ul ekleyerek, 10 mg / ml&#39;de bir amin-reaktif floresan boyalar (AlexaFluor 532-K) içinde çözülür. vorteks ile karıştırın. Kullanımdan hemen önce bu çözüm olun. </li><li> antikorun, 100 ug amin-reaktif boya 2.5 ul ekle. Not: Genellikle reaktif boyanın 5-20 mol fazlası önerilir. Bununla birlikte, reaktif boyanın miktarı, reaksiyon ampirik antikor oranı ve içsel antikor özellikleri arzu edilen boya bağlı olarak optimize edilmesi gerekmektedir ilave edildi. </li><li> vorteksli bir mikserde veya son aşırı uç mikser kullanılarak, oda sıcaklığında 60 dakika boyunca örnek karıştırın. boncuk suspensio kalır emin olunn. </li><li> 10 saniye boyunca manyetik stand tüp yerleştirin ve süpernatant kaldırmak. </li><li> antikor bağlama / yıkama tamponu 250 ul ilave et ve karıştır. 10 saniye boyunca manyetik stand koyun. Çıkarın ve bağlayıcı / yıkama tamponu atın. İki yıkar toplam için bu adımı tekrarlayın. </li></ol></li><li> antikor Kurtarma <ol><li> tanelerine elüsyon tamponu 50 ul ekle. </li><li> vorteksli bir mikserde veya son aşırı uç mikser kullanılarak oda sıcaklığında 5 dakika boyunca karıştırın. boncuk süspansiyon kalır emin olun. </li><li> 10 saniye boyunca manyetik stand tüp yerleştirin. nötralizasyon tamponu 5 ul içeren yeni bir mikro-santrifüj tüpüne yıkandı örnek ve transfer çıkarın. </li><li> sürecini bir kez daha tekrarlayın ve yıkandı, örnekleri havuz. </li><li> antikor konsantrasyonunu ölçmek ve boya için antikor oranı Bölüm 4&#39;te tarif edildiği gibi. </li></ol></li></ol> Tiol kimyası kullanılarak 3. Antikor etiketleme <ol><li> antikor Azaltma <ol><li> 250 ul ekleyintiyol konjugasyon tampon ve karışımı. 10 saniye boyunca manyetik stand tüp yerleştirin. Çıkarın ve tampon atın. İki kez bu adımı yineleyin. </li><li> tiyol konjugasyon tampon 100 ul ekleyin. </li><li> 2.5 mM&#39;lik bir son konsantrasyona ditiyotreitol (DTT) ilave edilir. </li><li> vorteksli bir mikserde veya son aşırı uç mikser kullanılarak, oda sıcaklığında 60 dakika boyunca örnek karıştırın. boncuk süspansiyon kalır emin olun. </li><li> 10 saniye boyunca manyetik stand tüp yerleştirin ve tampon atın. </li><li> tiyol konjugasyon tampon 250 ul ekleyin ve karıştırın. 10 saniye boyunca manyetik stand tüp yerleştirin. Çıkarın ve tampon atın. İki yıkar toplam için bu adımı tekrarlayın. </li><li> tiyol konjugasyon tampon 100 ul ekleyin. </li></ol></li><li> Konjuge Antikor <ol><li> boyanın 1.0 mg DMSO, 100 ul ekleyerek / ml 10 mg tiol-reaktif boya çözülür. vorteks ile karıştırın. Kullanımdan hemen önce bu çözüm olun. </li><li> anti 100 ug için tiol-reaktif boya 2.5 ul eklevücut. Not: Genellikle reaktif boyanın 5-20 mol fazlası önerilir. Bununla birlikte, reaktif boyanın miktarı, reaksiyon ampirik antikor oranı ve içsel antikor özellikleri arzu edilen boya bağlı olarak optimize edilmesi gerekmektedir ilave edildi. </li><li> vorteksli bir mikserde veya son aşırı uç mikser kullanılarak, oda sıcaklığında 60 dakika boyunca örnek karıştırın. boncuk süspansiyon kalır emin olun. </li><li> 10 saniye boyunca manyetik stand tüp yerleştirin ve süpernatant kaldırmak. </li><li> tiyol konjugasyon tampon 250 ul ekleyin ve karıştırın. 10 saniye boyunca manyetik stand koyun ve tampon kaldırmak. İki yıkar toplam için bu adımı tekrarlayın. </li></ol></li><li> Zehir Antikor <ol><li> tanelerine elüsyon tamponu 50 ul ekle. </li><li> vorteksli bir mikserde veya son aşırı uç mikser kullanılarak oda sıcaklığında 5 dakika boyunca karıştırın. boncuk süspansiyon kalır emin olun. </li><li> 10 saniye boyunca manyetik stand tüp yerleştirin. neut 5 ul içeren yeni bir mikro-santrifüj tüpüne yıkandı örnek ve transfer çıkarınralization tamponu. </li><li> sürecini bir kez daha tekrarlayın ve yıkandı, örnekleri havuz. </li><li> antikor konsantrasyonunu ölçmek ve boya için antikor oranı Bölüm 4&#39;te tarif edildiği gibi. </li></ol></li></ol> 4. Hesapla Dye-to-Antikor Oranı <ol><li> 280 nm (A280) de ve boya (Amak) için Xmaks antikor boya birleşimine absorbansı ölçülür. </li><li> antikor konsantrasyonunu hesaplayın: Antikor konsantrasyonu (mg / ml) = A280 - (CF x A max) / 1.4 nerede CF (üretici tarafından sağlanan) boya = düzeltme faktörü. </li><li> boya-to-antikora oranı hesaplanır: Boya-to-antikor oranı (DAR) = (150,000 x A max) / Ab konsantrasyonu (mg / ml) x ε boyası burada, ε boya = sönüm absorbansı en fazla boyanın katsayısı ve antikorun molekül ağırlığı = 150.000 Da. </li></ol>

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Authors are employees of Promega Corporation.

Bu çalışmanın amacı, küçük moleküllerin çeşitli, düşük konsantrasyonlarda, hücre ortamı içinde mevcut antikorları etiket için bir yöntem geliştirmektir. Antikor keşif erken safhalarında antikorların çok sayıda olanak sağlayacaktır bir yöntem olup, küçük moleküller ile işaretlenmiş olan bir biyolojik ilgili deneyi kullanılarak taranabilir için. Böyle bir deney, içselleştirme da benzer bağlanma afinitelerine sahip antikorlar arasında değişebilir reseptör aracılı antikor, geli...

Küçük moleküller ile etiketli antikorlar, belki de biyoloji 1,2 en yaygın olarak kullanılan reaktiflerdir. Floresan boyalar ve biyotin ile etiketlenmiş antikorlar yoğun diğer uygulamalar 3-6 arasında sitometrisi Western blot ve immüno-çökeltme, akış görüntüleme, bağışıklık kullanılır. Radyoaktif işaretli antikorlar 3,7 kanserlerin tedavisi için yeni seçenekler sunuyoruz, ve iki ADC zaten terapötik kullanım 8 için onaylanmıştır görüntüleme ve tedavi, sitotoksik ilaçlar (ADK) ile etiketlenmiş antikorlar yaygın kullanım bulmaktadır. Bunların yaygın kullanımı olmasına rağmen, etiketleme antikorlar için yöntemler şaşırtıcı değişmemiş ve tipik olarak çok sayıda reaksiyonlar içerir ve tuz giderme 9-12 aşama vardır. Çözüm yöntemleri sadece birkaç antikorlar etiketlenmesi gerekir ve yüksek konsantrasyonda yüksek arıtılmış formda mevcut olduğu durumlarda ve yeterli hacimlerde çok iyi çalışmaz. Ancak, ADK gibi yeni uygulamalar için, orada birBunlar, örneğin, reseptör aracılı antikor, geliştirilmiş içselleştirme 13-16 için, biyolojik olarak uygun özellikler için taranabilir erken böylece hibridom aşamasında antikorlar etiket gerekir. hibridom aşamada, numune hacimleri antikorlar düşük konsantrasyonlarda ifade numune sayısı dolayısıyla çözelti bazlı etiketleme yöntemleri uygun değildir, büyük olan, sınırlıdır. Basitleştirmek ve geleneksel antikor etiketleme yöntemleri verimini artırmak için, birkaç alternatif yaklaşımlar 17,18 önerilmiştir. Bir yaklaşım, etiketleme reaksiyonu ve etiketlenmiş, saflaştırılmış protein tasfiye edilir antikorları yakalamak için manyetik olmayan Proteini küçük sütunlarda paketlenmiş bir afinite taneleri kullanmaktır. Bu yöntem, hücre ortamı şirketinden antikorları etiketlemek için kullanılabilir, ancak sütun kullanımı zahmetli olabilir. Bir manyetik boncuk tabanlı bir yöntem son zamanlarda sütun kullanımını ortadan kaldırır ve hacmini fakat geliştirir 19 rapor edilmiştirboncuk bağlama kapasitesi, sınırlı antikora, alçak antikor mikrogram düzeylerindeki miktarlarda sadece nanogram etiketlenmiş olabilir. Biz son zamanlarda geliştirilen ve yüksek kapasitesi manyetik Protein A ve Protein G boncuk (&gt; İnsan IgG / yerleşik boncuk ml 20 mg) küçük moleküller 20 hücre medyada mevcut antikorları etiketlemek için kullanılır. tanelerin yüksek kapasiteli antikorun mikrogram On ila yüzlerce uygun etiketlenmesine izin verir ve boncuk hızlı bir manyetik müdahale paralel örneklerin çok sayıda taşıma ve işleme kolaylaştırır. Küçük moleküller için belirteç gibi floresan boyalar kullanarak, yöntem, bir amin ve tiyol markalama kimya ile uyumlu ve etiketlenmiş ve çok saf antikorların yüksek geri kazanımlar sahip olduğunu gösteriyor. Bu protokol ve beraberindeki bir video Manyetik Protein A ve Protein G boncuklar kullanılarak hücre medyada mevcut fare antikorları üzerinde boncuk etiketleme açıklar. protokoldürdört bölüme ayrılır: Bölüm 1 biyolojik örneklerden boncuk üzerine antikorların fethini şöyle anlatır. yakalanmasını takiben, amin kimyası kullanılarak veya tiyol kimya kullanılarak floresan boya ile antikor etiketleme sırasıyla 2 ve 3. bölümde tarif edilmektedir. Son olarak, bölüm 4 antikoru konsantrasyonu hesaplamalar ve antikor oranı boya için bir yöntem açıklanır.

<table><tbody><tr><td>Magne Protein A Beads</td><td>Promega Corporation</td><td>&amp;nbsp;G8781</td><td></td></tr><tr><td>Magne Protein G Beads</td><td>Promega Corporation&amp;nbsp;</td><td>&amp;nbsp;G7471</td><td></td></tr><tr><td>AlexaFluor 532-SE (Succinimidyl Ester)</td><td>Life Technologies</td><td>A20001</td><td></td></tr><tr><td>AlexaFluor 532-ME (Malemide)</td><td>Life Technologies</td><td>A10255</td><td></td></tr><tr><td>AlexaFluor 647-ME (Maleimide)</td><td>Life Technologies</td><td>A20347</td><td></td></tr><tr><td>Fluorescein-ME (Maleimide)</td><td>Life Technologies</td><td>F-150</td><td></td></tr><tr><td>Magnetic Stand</td><td>Promega</td><td>Z5332</td><td></td></tr></tbody></table>

antibody labeling with fluorescent dyes using magnetic protein a and protein g beads

Floresan boyalar, sitotoksik ilaçlar ve radyoaktif iz gibi küçük moleküller ile işaretlenmiş olan antikorlar, esas biyomedikal araştırmalarda araçları, bağışıklıkla ilgili tanı ve daha yakın olarak terapötik maddelerdir. küçük moleküller ile markalama antikorlar için geleneksel yöntemler, nispeten yüksek bir konsantrasyonda antikor saflaştınldı birden diyaliz adımları içerir ve veri akışını sınırlı gerektirir. Ancak, Antikor İlaç Eşlenikleri (ADC) alanında olmak üzere çeşitli uygulamalar, hücre ortamından doğrudan antikorların etiketleme sağlayacak yeni yöntemler yararlanacaktır. Bu tür yöntemler antikorlar örneğin, biyolojik olarak uygun deneylerde taranabilir izin verebilir, ADC&#39;ler halinde reseptör aracılı antikor, geliştirilmiş içselleştirme deneyi. Burada, doğrudan hücre ortamından antikorlar küçük miktarlarda etiketleme sağlayan bir yöntem olup (on-boncuk yöntemi) tarif eder. Bu yaklaşım antikorları yakalamak için yüksek kapasiteli manyetik Protein A ve Protein G afinite boncuklar kullanıramin veya tiyol kimya ve etiketlenmiş antikorlar daha sonra elüsyon kullanılarak küçük moleküller ile etiketleme ve ardından hücre ortamından. küçük moleküller için suretler olarak floresan boyalar alırsak, biz doğrudan amin ve tiyol etiketleme kimyası her ikisini de kullanarak hücre medyadan üç farklı fare antikorları on-boncuk etiketleme göstermektedir. Protein A ve Protein G antikorların yüksek bağlanma afinitesi, yüksek geri kazanımlar olarak etiketlenmiş antikorların yüksek saflıkta sağlar. Buna ek olarak, manyetik boncuk kullanımı çoklu numuneler önemli oranda markalama verimliliği arttırır, elle kullanılmasına izin verir.

Yüksek kapasiteli manyetik Protein A ve Protein G boncuklar kullanılarak küçük moleküller ile antikor etiketleme için bir şeması, Şekil 1 &#39;de gösterilmiştir. Antikorlar etiketler amin kimyası kullanılarak, örneğin flüoresan boyalar, G boncuklar küçük moleküller ile etiketlenebilir manyetik Protein çekilen lizin amino asitler ya da antikor menteşe bölgesindeki düşük tiyoller etiketler, tiol kimya kullanılarak birincil aminlerdir. Antikorlar...

Watch this Scientific Journal Video about Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads at JoVE.com

Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

küçük moleküller ile antikorlar için etiketleme üzerine boncuk yöntem doğrudan hücre ortamından antikorlar, az miktarda etiketleme sağlar. Bu yöntem amin ve tiyol kimyası ile uyumlu ve paralel olarak çoklu örnekleri, elle veya otomatik platformları kullanarak işleyebilir.

Floresan Boyalar ile Antikor Etiketleme Manyetik Protein A ve Protein G Boncuk kullanma

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, ...

antibody-labeling-with-fluorescent-dyes-using-magnetic-protein

Research

JoVE Journal

Immunology and Infection

11.8K Görüntüleme.  Promega Corporation.  küçük moleküller ile antikorlar için etiketleme üzerine boncuk yöntem doğrudan hücre ortamından antikorlar, az miktarda etiketleme sağlar. Bu yöntem amin ve tiyol kimyası ile uyumlu ve paralel olarak çoklu örnekleri, elle veya otomatik platformları kullanarak işleyebilir. 

Video: Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is protein conjugation by genetically introducing an unnatural amino acid containing a bioorthogonal functional group. This report describes a detailed protocol for site-specific antibody conjugation. The protocol includes experimental details for the genetic incorporation of an azide-containing amino acid, and the conjugation reaction by strain-promoted azide-alkyne cycloaddition (SPAAC). This strain-promoted reaction proceeds by simple mixing of the reacting molecules at physiological pH and temperature, and does not require additional reagents such as copper(I) ions and copper-chelating ligands. Therefore, this method would be useful for general protein conjugation and development of antibody drug conjugates (ADCs).

Here, we present a protocol to site-specifically introduce chemical probes into an antibody fragment by genetically incorporating an azide-containing amino acid, and subsequently coupling the azide with a chemical probe by strain-promoted azide-alkyne cycloaddition (SPAAC).

Verimli ve Siteye özgü Gerilme-terfi Azid-alkin Siklokatılma tarafından Antikor Etiketleme

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is ...

Biyokimya

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has relevant health and economic consequences. Many cyanobacterial strains are toxin producers, and only a few cells are necessary to induce irreparable damage to the environment. Therefore, water-body authorities and administrations require rapid and efficient early-warning systems providing reliable data to support their preventive or curative decisions. This manuscript reports an experimental protocol for the in-field detection of toxin-producing cyanobacterial strains by using an antibody microarray chip with 17 antibodies (Abs) with taxonomic resolution (CYANOCHIP). Here, a multiplex fluorescent sandwich microarray immunoassay (FSMI) for the simultaneous monitoring of 17 cyanobacterial strains frequently found blooming in freshwater ecosystems, some of them toxin producers, is described. A microarray with multiple identical replicates (up to 24) of the CYANOCHIP was printed onto a single microscope slide to simultaneously test a similar number of samples. Liquid samples can be tested either by direct incubation with the antibodies (Abs) or after cell concentration by filtration through a 1- to 3-&#956;m filter. Solid samples, such as sediments and ground rocks, are first homogenized and dispersed by a hand-held ultrasonicator in an incubation buffer. They are then filtered (5 - 20 &#956;m) to remove the coarse material, and the filtrate is incubated with Abs. Immunoreactions are revealed by a final incubation with a mixture of the 17 fluorescence-labeled Abs and are read by a portable fluorescence detector. The whole process takes around 3 h, most of it corresponding to two 1-h periods of incubation. The output is an image, where bright spots correspond to the positive detection of cyanobacterial markers.

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.

Saptamak için deneysel protokol<em&gt; Siyanobakteri</em&gt; Bir Antikor Mikroarray Chip ile Sıvı ve Katı Örneklerinde

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has ...

Çevre

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

Canlı Kültür Nöronlar Antikor Besleme sonra Hücre-yüzey ve İçselleştirilmiş Proteinlerin Diferansiyel Etiketleme

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

Nörobilim

Magnetic Tweezers in a Microplate Format

Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these approaches are limited single-molecule methods, which restricts their availability. To address this need, a microplate assay was developed that enables mechanical manipulation while performing standard biochemical assays. This is achieved using magnets incorporated into a microplate lid to create multiple magnetic tweezers. In this format, force is exerted across biomolecules connected to paramagnetic beads, equivalent to a typical magnetic tweezer. The study demonstrates the application of this tool with FRET-based assays to monitor protein conformations. However, this approach is widely applicable to different biological systems ranging from measuring enzymatic activity through to the activation of signaling pathways in live cells.

Here, we describe the use of a novel microplate assay to enable mechanical manipulation of biomolecules while performing ensemble biochemical assays. This is achieved using a microplate lid modified with magnets to create multiple static magnetic tweezers across the microplate.

Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

Dayanıksız Multi-protein Kompleksleri izolasyonu In vivo Kontrollü Hücresel Çapraz Bağlama ve immuno-manyetik Afinite Kromatografisi

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Biyoloji

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR-Klip - RNA Cilt Proteinler Transcriptome geniş Cilt Siteleri Belirlemek Amacıyla Bir Yöntem

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

Elektron Spektroskopik Görüntüleme Kombinasyonunun miniSOG Etiketli DNA Onarım Proteinler Görselleştirme (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

Fare Karaciğerinde Hücre Tipine Özgü Gen Ekspresyonu Profiloluşturma

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.

We here introduce a procedure to measure protein oligomers and aggregation in cell lysate and live cells using fluorescence correlation spectroscopy.

Floresan Korelasyon Spektroskopisi Kullanılarak Protein Toplamanın Tespiti

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's ...

A Technique to Detect Cyanobacterial Strains Using an Antibody Microarray Chip

Source: Blanco, Y. et al., <a href="https://app.jove.com/v/54994">Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates a method to detect the Cyanobacterial strains in an environmental sample using an antibody microarray chip. Samples carrying various bacteria interact with printed antibodies, and immunostaining with fluorophore-labeled anti-cyanobacterial-strain antibodies reveals the presence of specific cyanobacterial strains through fluorescence in distinct spots.

Floresan Boyalar ile Antikor Etiketleme Manyetik Protein A ve Protein G Boncuk kullanma

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