Yazarlar radyoaktif etiketli virüs hazırlanması için Farmasötik Biyoteknoloji ve Moleküler Biyoloji Bölümü ve özellikle Monique De Pelsmacker personeline teşekkür ederim. Biz Ellen Merckx ve onların ilginç açıklamalar ve tartışmalar için Hadewych Halewyck ve laboratuvarda yaptığı yardım Gerrit De Bleeser minnettarız. Bu çalışma, maddi Vrije Universiteit Brussel (OZR1807), Fonds voor Wetenschappelijk Onderzoek Vlaanderen (G.0168.10N) ve Dünya Sağlık Örgütü (TSA 200.410.791) bir OZR hibe ile desteklenmiştir. Lise Schotte Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO) bir predoctoral üyesidir.

Prensibi (A) ve yöntemi genel bir (B) Şekil 1 içinde tasvir edilmiştir. 1. Tampon hazırlanması <ol><li> Erime sodyum dihidrojen fosfat (50 mM) ve su içinde sodyum klorür (300 mM) ile Yıkama / bağlanma tampon hazırlamak ve 8.0 pH ayarlanır. Buna ek olarak metiyonin (% 2 (m / v) son konsantrasyon) ve albumin Tween 20 (% 0.01 (m / v) son konsantrasyon), (0.1% (m / v) son konsantrasyon) ekleyin ve gerekli hacmi e ayarlanır. </li></ol> 2. Manyetik Boncuk hazırlanması Kılavuzuna göre manyetik boncuk hazırlayın. Kısaca: <ol><li> Bir girdap kullanarak manyetik boncuk süspansiyon hale getirin. </li><li> Transferi, her bir örnek için, bir tüp içine yeniden süspanse manyetik boncuklar (40 mg / ml) 10 ul. </li><li> Süpernatant netleşene kadar, bir mıknatıs kullanarak manyetik boncuk toplayın (+ / - 30 sn) ve supernatant dikkatlice çıkarın. </li><li> Yıkayınmanyetik boncuk iki kez: Yıkama / Bağlama tampon 150 ul içinde boncuklar tekrar süspansiyon. Manyetik boncuk süpernatant netleşene kadar bir mıknatıs kullanarak tüpün bir tarafına göç ve dikkatlice supernatant çıkarın. </li><li> Boncuklar (10 mg / ml) tekrar süspansiyon için, her bir örnek için, Yıkama / bağlanma tampon maddesi 40 ul kullanabilir. </li></ol> 3. Affinity Yakalama Testi Not: (kozmik) arka radyoaktivite (0% radyoaktivite) ölçmek için, herhangi bir radyoetiketli virüsü bir kontrol örneği 1 eklenir. Bunun yerine, aynı hacimde Binding / Yıkama tamponu ilave edilir. Not: 100% radyoaktivite, tüm bileşenler nanobody hariç eklenir için bir kontrol örneği 2 ile tanımlanır. Bunun yerine, aynı hacimde Binding / Yıkama tamponu ilave edilir. <ol><li> Bir 96-kuyucuklu mikrotitre plakası içine 80 ul kadar Yıkama / bağlanma tampon maddesi ile seyreltildi (β-radyasyon) poliovirus Sabin suşu tipi 1 9, 35 etiketli S 2000 cpm getirir. </li><li> Ekle 10 &amp; mu; kuyulara nanobody seyreltme l. </li><li> Bir çalkalayıcı kullanarak yaklaşık 10 saniye karıştırın. </li><li> Numuneler oda sıcaklığında 1 saat inkübe izin verin. </li><li> Yıkanmış manyetik boncuklar süspansiyonu, 40 ul ekleyin ve sürekli çalkalama altında, oda sıcaklığında 10 dakika süreyle inkübe edilir. </li><li> Boncuk ve bir mıknatıs kullanarak süpernatant ayırın ve bir tüp içine temizlenmiş süpernatant aktarın. </li></ol> 4. Radyoaktivite ölçümü <ol><li> Bir sayma balona adım 3.6 den süpernatan 50 ul aktarın. </li><li> Sintilasyon sıvısı ve karışım 3 ml ilave edilir. Bir β-sintilasyon sayacında radyoaktivite ölçün. </li></ol> 5.. Boncuk Geri Dönüşüm <ol><li> 2 dakika boyunca 500 xg&#39;de veya açık bir süpernatant elde edilinceye kadar bir tüp ve santrifüj kullanılan manyetik boncuk toplayın. </li><li> Süpernatantı ve 6 ml 0.5 M NaOH içinde boncuklar tekrar süspansiyon. </li><li> Multipl için süspansiyon aktarıne tüpler ve 5 dakika boyunca bir ultrason banyosunda inkübe. </li><li> Manyetik boncuk toplamak ve süpernatant kaldırmak için mıknatıslar kullanın. </li><li> Her tüpe 1 ml% 2 SDS-çözüm ekleyin ve bir girdap kullanarak tekrar süspansiyon. 5 dakika boyunca kaynatın. </li><li> Boncuklar toplayın ve supernatant çıkarın. 1 ml 0.2 M EDTA (pH = 7) içerisinde yeniden süspanse boncuklar. </li><li> 5 dakika boyunca bir ultrason banyosunda tüpler ve bir kez daha inkübe, süpernatan kaldırın. </li><li> Her tüpe 1 mL su ekleyin ve boncuklar tekrar süspansiyon. Boncuklar toplayın ve supernatant çıkarın. Iki kez bu yıkama işlemi tekrarlayın. </li><li> Süpernatan kaldırmak ve 1 ml 10 mM CoCl 2 çözelti içinde yeniden süspanse boncuklar. 10 dakika bir çalkalayıcı koyun. </li><li> Boncuk toplamak ve Yıkama / Ciltleme tampon 1 ml tekrar süspansiyon için bir mıknatıs kullanarak süpernatantı. </li><li> Süpernatant kaldırmak ve boncuklar 20% 1 ml kullanılarak iki kere (v / v) etanol yıkayıp </li><li> % 20 kendi orijinal hacim (v / v) ve in boncuklar tekrar süspansiyon40 mg / ml &#39;lik bir konsantrasyonda yeniden elde etmek için boncuklar hanol. </li></ol> 6. Sonuçların Yorumlanması <ol><li> Hiçbir radyoaktif virüs eklenmiş olan Kontrol numunesi 1, arka radyoaktivite düzeltmek için ve% 0 radyoaktivite değeri ayarlamak için kullanılır. Nanobody nerede ve dolayısıyla tüm radyoaktif virüs süpernatant kalır içermez Kontrol örnek 2,% 100 radyoaktivite değeri olarak ayarlanır. </li><li> Nanobody tarafından radyoaktif virüsün - süpernatan radyoaktivite yüzdesi (= a%) toplam yağış (a% = 100) bir ölçüsüdür. </li></ol> 7. Temsilcisi Sonuçlar Bu afinite yakalama testinin sonuçları temsili Şekiller 2, 3 ve 4 &#39;de gösterilmiştir. Şekil 2&#39;de, PVSS38C, poliovirus için bir nanobody spesifik afinite ile gösterilir. PVSS38C bir afinite poliov üç farklı antijenleri için test edildiirus: Yerli-antijen (N-antijen), Isıtmalı-antijen (H-antijen) ve 14S alt birimleri. N-antijen bulaşıcı intakt bir virüstür. Virüs ısıtılır (56 azından 20 dakika ° C) ya da viral RNA&#39;nın bir (kısmi) kaybı ve kapsid proteini VP4 ile sonuçlanan bir katı yüzey (yayınlanmamış sonuçlar), kapsid değişikliklerin konformasyon, ekli ve boş olduğunda capsids sonra H-antijen olarak adlandırılan edildiği oluşturulmaktadır. 14S alt birimleri montaj ara vardır. Bu antijenler, aşı sonrası 10 antikorların farklı setleri tarafından tanınan ve bu nedenle farklı epitopları ve antijenik sitelere sahip edilir. Şekildeki süpernatan bulunan radyoaktivite yüzdesi nanobody konsantrasyonunun bir fonksiyonu olarak temsil edilir. Bu radyoetiketli 14S ait birimlerini içeren örneklerin üstte yüzen madde içindeki radyoaktivite nanobody PVSS38C artan konsantrasyonları ile azalmaktadır olduğu görülebilir. Bu poliovirus 14S alt birimleri con miktarında bir artış olarak tercüme edilebilirnanobody PVSS38C ve dolaylı manyetik boncuk bağlanırsınız. Yakalama titresi (radyoaktif virüs% 50 yakalama nanobody gerekli of = konsantrasyon) grafiksel 0.099 nM olarak hesaplanabilir. N-antijenik ve poliovirus H-antijenik formu için PVSS38C No afinite görülmektedir. Bu verilere bakıldığında PVSS38C poliovirus diğer iki antijenik formları sadece 14S alt birim mevcut değildir ve bir epitopu ile etkileşim olduğunu yorumlanır. Süpernatan gelen radyoaktivite kaybı sadece antijenleri doğru nanobody spesifik afinite bağlı olduğunu göstermek için, aynı deney Ab1 ile gerçekleştirildi, Sulfolobus sulfataricus arasında lrpB transkripsiyon faktörü karşı oluşturulan ve bilinen bir nanobody bir etkileşim ile sahip Herhangi bir poliovirus antijen. Bu değerlendirmenin sonucu, Şekil 3 &#39;de gösterilmiştir. Bütün radyoetiketli virüsünün herhangi reaktivitesi olduğunu gösteren süpernatanlar kalırPoliovirus üç antijenik türlerine karşı Ab1. Sonuçların tekrarlanabilirliği farklı günde bir verilen nanobody (yani, PVSP29F) sekiz defa olmak üzere toplam deneyi tekrarlayarak ve farklı kişiler tarafından test edildi. Sonuçlar Şekil 4 &#39;de gösterilmiştir. Süpernatant yüzde radyoaktivitenin ortalama değeri her nanobody konsantrasyonu için hesaplandı ve standart sapma ile yazışmalarda temsil edilmektedir. <img alt="Şekil 1.." src="/files/ftp_upload/3937/3937fig1.jpg" /> Şekil 1.. Assay of prensibi (A) bir özetini ve (b) yöntemi. A. özellikle belli bir poliovirus antijen kobalt kaplı manyetik bilye ile etkileşim ve radyoaktif işaretli virüs çöktürmek olacaktır mümkün olacak tanımak nanobodies Onun etiketli. B. His-etiketli nanobodies radyoaktif olarak etiketlenmiş poliovirus eklenir ve inkübe edilir. Kobalt-kaplı manyetik bilye vardıreklenmiş ve ilişkili / ilişkisiz antijen manyetik ayırma gerçekleştirilir. Üstte kalan radyoaktivite ölçülür ve ele antijen miktarda elde edilebilir. <img alt="Şekil 2" src="/files/ftp_upload/3937/3937fig2.jpg" /> Şekil 2. Nanobody PVSS38C farklı poliovirus antijenlerin Manyetik boncuk afinitesi yakalama. Süpernatan bulunan radyoaktivite yüzdesi PVSS38C konsantrasyonunun bir fonksiyonu olarak temsil edilir. 14S için bir konsantrasyon-yanıt ilişkisi 14S münhasıran mevcut bir epitopu ile PVSS38C etkileşimi gösteren bulunabilir. Ile belirgin bir etkileşim N-, ne de bir non-spesifik çok yüksek konsantrasyonlarda yakalama ihmal edilebilir bir fark edilmiş olmasına rağmen H-antijen, tespit edilebilir. <img alt="Şekil 3" src="/files/ftp_upload/3937/3937fig3.jpg" /> Şekil 3. Nanobody Ab1 farklı poliovirus antijenlerin Manyetik boncuk afinitesi yakalama </strong&gt;. Süpernatan bulunan radyoaktivite yüzdesi Ab1 konsantrasyonunun bir fonksiyonu olarak temsil edilir. Ab1 herhangi poliovirus-antijen ya-alt birimi olan bir etkileşim olduğu bilinmektedir. Radyoaktivitenin% 100 süpernatant bulunan bu yana, hiçbir virüs non-spesifik Ab1 bağlı idi. Şekil 2&#39;de radyoaktivite kaybı dolayısıyla antijenleri doğru nanobody spesifik afinite sadece kaynaklanmaktadır. <img alt="Şekil 4." src="/files/ftp_upload/3937/3937fig4.jpg" /> Şekil 4. Testin tekrarlanabilirliği. Üstte yüzen madde içindeki yüzdesi radyoaktivite ortalama değeri nanobody konsantrasyonunun bir fonksiyonu olarak temsil edilir. Deney, farklı gün ve farklı kişiler tarafından sekiz kez tekrar edildi. Standart sapma dikey çubuklar olarak temsil edilir.

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Biz ifşa hiçbir şey yok.

Protokolde, nanobody ile etkileşen antijen radyoaktivite süpernatan gelen radyoetiketli virüsünün kaybı olarak tanımlanmaktadır. Bu nedenle çöken radyoaktivite (= 100 -%) miktarı (manyetik boncuk bağlı) supernatant (= bir%) olarak radyoaktivite tarafından dolaylı bir şekilde tahmin edilebilir. Diğer taraftan, 500 mM imidazol ile manyetik boncuklar immün elüt tarafından doğrudan bir şekilde antijen çöktürülmüş bağlı fraksiyonun radyoaktivite ölçmek için de mümkündür. Bu, daha önce rad...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strongReaktifin&gt; Adı</strong</td&gt;<td<strong&gt; Şirket</strong</td&gt;<td<strong&gt; Katalog numarası</strong</td&gt;<td<strong&gt; Yorumlar</strong</td&gt;</tr&gt;<tr&gt;<td&gt; Dynabeads His-Tag İzolasyon ve Pulldown</td&gt;<td&gt; Invitrogen</td&gt;<td&gt; 101.03D</td&gt;<td&gt; Manyetik boncuk</td&gt;</tr&gt;<tr&gt;<td&gt; Optiphase &#39;HiSafe&#39; 2</td&gt;<td&gt; Perkin Elmer</td&gt;<td&gt; 1200 - 436</td&gt;<td&gt; Sintilasyon sıvısı</td&gt;</tr&gt;</tbody&gt;</table

a liquid phase affinity capture assay using magnetic beads to study protein-protein interaction: the poliovirus-nanobody example

Bu makalede, basit, kantitatif, sıvı faz afinitesi yakalama deneyi sunulmuştur. Bir proteinin etiketlenmiş edilebilir olması şartıyla ve başka protein etiketli, bu yöntem, protein-protein etkileşimlerinin araştırılması için de uygulanabilir. Bu, kobalt kaplı manyetik boncuklar ile etiketlenmiş protein tanınması ile etiketlenmiş protein ve etiketlenmiştir ikinci bir özel protein arasındaki etkileşimi, diğer yandan, bir taraftan dayanır. İlk olarak, etiketlenmiş ve etiketlenmiş proteinleri karıştırılır ve oda sıcaklığında inkübe edilir. Etiketi kabul manyetik boncuk, eklenir ve etiketli protein bağlı fraksiyonu mıknatıslar kullanılarak ilişkisiz fraksiyon ayrılır. Yakalanır etiketli protein miktarı, ilişkisiz fraksiyonu içinde kaldı etiketli protein sinyali ölçülmesi ile dolaylı bir şekilde belirlenebilir. Yapısal dönüşüm duyarlı proteinler eşek olduğunuzda açıklanan sıvı faz afinitesi tayini son derece yararlıayed. Assay of gelişimi ve uygulama poliovirus ve nanobodies 1 tanıyan poliovirus arasındaki etkileşim için gösterilmiştir. Poliovirus katı bir yüzey (yayınlanmamış sonuçlar) eklenmiş konformasyonel dönüşüm 2 karşı hassas olduğu için, ELISA kullanımı sınırlıdır ve bir sıvı faz bazlı sistemi nedenle tercih edilmelidir. Genellikle polioresearch 3,4 kullanılan bir sıvı fazda tabanlı bir sistemin bir örneğini mikro protein A-immünopresipitasyon testi 5 &#39;dir. Bu test uygulanabilirliğini kanıtlamış olsa da, 6,7 nanobodies bulunmaz bir Fc-yapısı gerektirir. Ancak, başka bir fırsat olarak, bu ilginç ve istikrarlı tek alan antikorlar 8 kolayca farklı etiketler ile tasarlanmış olabilir. Yaygın (O&#39;nun) 6-etiketi gibi onların dönüşünü kolaylıkla manyetik boncuk kaplı edilebilir nikel ve kobalt gibi iki değerlikli iyon afinite gösterir kullanılır. Bu nedenle, bu basit kantitatif geliştirdiafinite yakalama testi kobalt kaplı manyetik bilye dayalı. Poliovirüs nanobodies ile engellenmeden etkileşimi etkinleştirebilirsiniz ve nicel bir saptama yapılabilir kılmak için 35 S etiketli edildi. Metodu gerçekleştirmek için kolaydır ve daha etkili bir şekilde yenileyici manyetik boncuklar olasılığını tarafından desteklenen bir düşük maliyet ile tespit edilebilir.

Watch this Scientific Journal Video about A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example at JoVE.com

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

Bu makalede, basit, kantitatif, sıvı faz afinitesi yakalama deneyi sunulmuştur. Bu bir yandan, diğer tarafta etiketli protein ve ikinci, etiketli protein (örn. poliovirus) arasındaki yakınlığı manyetik boncuk ve etiketli proteinlerin (örneğin nanobodies) arasındaki etkileşime dayanan güvenilir bir tekniktir.

Protein-Protein Etkileşimi Okumak Manyetik Boncuk Kullanarak Bir Sıvı Faz Affinity Yakalama Testi: Poliovirüs-Nanobody Örnek

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

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11.8K Görüntüleme.  Vrije Universiteit Brussel. Bu makalede, basit, kantitatif, sıvı faz afinitesi yakalama deneyi sunulmuştur. Bu bir yandan, diğer tarafta etiketli protein ve ikinci, etiketli protein (örn. poliovirus) arasındaki yakınlığı manyetik boncuk ve etiketli proteinlerin (örneğin nanobodies) arasındaki etkileşime dayanan güvenilir bir tekniktir.

Video: A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Sıvı Faz Kurtarma Moleküler Ağırlık Tabanlı Fraksiyonu GELFREE 8100 Fraksiyonu Sisteminin Kullanılması

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Biyoloji

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

Helios Gen Tabancası kullanarak Soğan Hücrelerde Protein-Protein Etkileşimi Bimoleküler Floresan tamamlama (BiFC) Testi

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Manyetik Aktif Hücre Ayırma (LIMACS) kullanarak Lipid veziküllü aracılı Afinite Kromatografisi: Protein-lipit Etkileşim Analiz Roman Yöntemi

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

Bir Multiplex Antikor Tarama Yöntemi kullanılarak bir Luminex xMAP Tahlil için bir Capture ELISA Dönüşüm

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

Bimoleküler Floresan tamamlama akışı Sitometrik Analizi: Protein-protein etkileşim Eğitim Bir Yüksek Verimli Kantitatif Yöntemi

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

Protein-protein etkileşimi belirlenmesi<em&gt; Drosophila</emTandem Affinity Arıtma tarafından&gt; Adult Başlar (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

Peptit Diziler protein-protein etkileşim Siteleri belirlenmesi

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron regulatory proteins, IRP1 and IRP2. They bind to iron responsive elements (IREs) within the untranslated regions (UTRs) of several target mRNAs, thereby controlling the mRNAs translation or stability. IRE/IRP interactions have been widely studied by EMSA. Here, we describe the EMSA protocol for analyzing the IRE-binding activity of IRP1 and IRP2, which can be generalized to assess the activity of other RNA-binding proteins as well. A crude protein lysate containing an RNA-binding protein, or a purified preparation of this protein, is incubated with an excess of32 P-labeled RNA probe, allowing for complex formation. Heparin is added to preclude non-specific protein to probe binding. Subsequently, the mixture is analyzed by non-denaturing electrophoresis on a polyacrylamide gel. The free probe migrates fast, while the RNA/protein complex exhibits retarded mobility; hence, the procedure is also called &#8220;gel retardation&#8221; or &#8220;bandshift&#8221; assay. After completion of the electrophoresis, the gel is dried and RNA/protein complexes, as well as free probe, are detected by autoradiography. The overall goal of the protocol is to detect and quantify IRE/IRP and other RNA/protein interactions. Moreover, EMSA can also be used to determine specificity, binding affinity, and stoichiometry of the RNA/protein interaction under investigation.

Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Here we present a protocol to analyze RNA/protein interactions. The electrophoretic mobility shift assay (EMSA) is based on the differential migration of RNA/protein complexes and free RNA during native gel electrophoresis. By using a radiolabeled RNA probe, RNA/protein complexes can be visualized by autoradiography.

IRE / IRP Örnek: RNA-protein Etkileşimleri Çalışmaları elektroforetik hareketlilik Shift Testi (EMSA)

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

Yaşayan Hücrelerde Protein-fragmanı tamamlama Assay (PCA) ile genom Protein-protein etkileşimi Eleme

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.