Name: analysis of histone antibody specificity with peptide microarrays
Uploaded: 2017-08-01T14:00:00.000+00:00
Duration: 9 min 47 s
Description: Watch this Scientific Journal Video about Analysis of Histone Antibody Specificity with Peptide Microarrays at JoVE.com

Bu çalışma kısmen Van Andel Araştırma Enstitüsü ve Ulusal Sağlık Enstitüsünden (CA181343) SBR&#39;e verilen bir araştırma ödeneği ile desteklendi

Yazarların açıklayacak bir şeyi yok.

Biyomedikal araştırma uygulamalarında antikor güvenilirliği en iyisi 46 , 47 . Bu, özellikle histamin PTM&#39;lerinin bolluğu ve dağılımını karakterize etmek için geliştirilen teknikler için anahtar araçlar olarak antikorların bulunduğu kromatin biyokimyasında geçerlidir. Burada sunulan protokol, histon PTM antikor özgünlüğünü analiz etmek için peptit mikrodizilerinin tasarım, imalat ve kullanımı için optimize edilmiş bir boru hat...

Genomik DNA, kromatin oluşturmak için ökaryotik hücre çekirdeğinin içinde histon proteinleri ile zarif bir şekilde paketlenmiştir. Kromatin tekrarlayan altbirimi, H2A, H2B, H3 ve H4 1 oktamerik bir çekirdek histon proteinleri etrafında sarılan 147 baz çift DNA&#39;dan oluşan nükleozomdur. Kromatin genel olarak gevşek biçimde paketlenmiş ökromatin ve yoğun sıkıştırılmış heterokromatin alanlar halinde düzenlenir. Kromatin sıkıştırma derecesi, protein makineleri, kopyalama, kopyalama ve onarım gibi temel DNA şablonlu işlemleri gerçekleştirmek için altta yatan DNA&#39;ya ne ölçüde erişebildiğini düzenler. Genetik erişilebilirliğin kromatin bağlamındaki kilit düzenleyicileri histon proteinlerinin yapılandırılmamış kuyruk ve çekirdek alanlarındaki 2 , 3 PTM&#39;leridir. Histon PTM&#39;leri doğrudan kromatin 4&#39;ün yapısını etkiler ve dolaylı olarak işlev görürlerh okuyucu proteinleri ve kromatin remodeling, enzimatik ve iskele aktivitelerini 5 sahip ilişkili makromoleküler kompleksler işe. Son iki on yılda histon PTM fonksiyonu üzerine yapılan çalışmalar, bu işaretlerin hücre akıbetini, organizma gelişimini ve hastalık başlatma / ilerlemesini düzenleyen önemli rol oynadığını göstermektedir. Kütle spektrometresi tabanlı proteomik teknolojideki ilerlemelerle beslenen 80&#39;den fazla farklı histon artıkları üzerinde 20&#39;den fazla benzersiz histon PTM&#39;si keşfedildi 6 . Özellikle, bu modifikasyonlar sıklıkla kombinasyon halinde ortaya çıkmakta ve &quot;histon kodu&quot; hipotezi ile tutarlı bir şekilde sayısız araştırma, histon PTM&#39;lerinin 7 , 8 , 9&#39;un spesifik kombinasyonlarının tanınması yoluyla okuyucu proteinlerinin farklı kromatin bölgelerini hedef aldığını göstermektedir. Önemli bir zorluk, işlevleri GrHistone PTM&#39;lerinin listesine bağlı olarak ve belirli histone PTM kombinasyonlarının kromatin ile ilişkili dinamik fonksiyonları nasıl düzenlediğini belirlemek için kullandı. Antikorlar, histon PTM&#39;lerinin tespiti için linç pinin reaktifleridir. Bu nedenle, kromatin biyokimya araştırmasında kullanılmak üzere 1.000&#39;den fazla histon PTM&#39;ye spesifik antikor ticari olarak geliştirilmiştir. Yüksek verimli DNA dizileme teknolojisinin hızlı gelişimi ile bu reaktifler, ChIP-seq&#39;deki bireysel araştırmacılar ve büyük ölçekli epigenomik &quot;yol haritası&quot; girişimleri ( örn. , ENCODE ve BLUEPRINT) tarafından geniş ölçüde kullanılmaktadır (kromatin immüno çökeltme, yeni nesil sıralama ) Histonun yüksek çözünürlüklü mekânsal haritalarını üretmek için boru hatları PTM dağıtım genomu genişliği 10 , 11 . Bununla birlikte, son çalışmalar histon PTM antikorlarının özgüllüğünün oldukça değişken olduğunu ve bu reaktiflerin unf<html> Hedef dışı epitop tanıma, komşu PTM&#39;ler tarafından güçlü pozitif ve negatif etki ve belirli bir kalıntı ( örn. , Mono-, di- veya tri-metillisin) 12 , 13 , 14 , 15&#39;deki modifikasyon sırasını ayırt etme zorluğu gibi avorable özellikler , 16 , 17 , 18 . Bu nedenle, bu değerli reaktiflerle üretilen verileri doğru bir şekilde yorumlamak için histon PTM&#39;ye özgü antikor reaktiflerinin titiz kalite kontrolü gereklidir. Mikroarray teknolojisi, binlerce makromoleküler etkileşimin aynı anda yüksek verimli, tekrarlanabilir ve minyatür formatta sorgulanmasını sağlar. Bu nedenle protein-DNA 19&#39;u analiz etmek için çeşitli mikrodizim platformları yaratılmıştır ,&quot;&gt; 20, protein-protein 21 ve protein-peptid etkileşimlerinin 22. Gerçekten de, histon peptid mikrodizileri yazarlar, silgi yüksek verimli profil sağlayan kromatin biyokimya araştırma için bilgi keşif platformu olarak ortaya çıktı ve histonun okuyucuları PTMS 15 adres , 23 , 24 ve ayrıca histone antikor özgünlüğünün analizi için 17 , 25. Histon peptit dizileri, kromatin ve epigenetik araştırmalarının ötesinde, sistemik lupus eritematosus ve diğer otoimmün hastalıkların teşhis / prognostik testi olarak potansiyel bir kullanıma sahiptir; kromatin otoantikorları, 27 26 oluşturulur. Burada, tasarım, imalat ve que için geliştirdiğimiz entegre bir boru hattı tanımlıyoruzHistonları ve PTM&#39;lerini tanıyan antikorlar için özgüllük profilleri üretmek için histon peptidi mikrodizileri kullandı. Boru hattı, yakın zamanda geliştirdiğimiz, mikroarray deneylerinin tasarım ve analiz aşamalarını bütünleştiren, açık kaynaklı ve interaktif bir yazılım uygulaması olan ArrayNinja tarafından kolaylaştırıldı 28 . ArrayNinja, Google Chrome&#39;da en iyi şekilde çalışır. Kısacası, streptavidin kaplı cam mikroskop lamlarında belirli bir konumda biyotinle konjuge edilmiş histone peptidlerinin bir kütüphanesinin depolanması için bir robotik temas mikrodizisi yazıcı kullanılır. Ardından diziler, antikor-epitop etkileşimlerini sorgulamak için rekabetçi ve paralel bir tahlil formunda kullanılabilir ( Şekil 1 ). Peptid kütüphanesi, tek başına PTM&#39;leri (lizin asetilasyonu, lisin / arginin metilasyonu ve serin / treonin fosforilasyonu) içeren yüzlerce benzersiz sentetik peptid ve büyük oranda proteomik veri kümelerinden türetilen ilgili kombinasyonlardan oluşur. Peptid sentezi ve geçerliliği için yöntemler<html> Başka yerlerde detaylandırılmıştır 23 . Bu dizilim platformunu kullanarak halen devam eden histon PTM antikor tarama çabalarımızdan elde edilen veriler, halka açık bir web kaynağı, Histone Antikor Özgüllilik Veritabanı (www.histoneantibodies.com) üzerinde arşivlenir. Özellikle, bu protokolün çeşitlemeleri ile imal edilen histon peptit mikroarrayleri, histon PTM okuyucu alanlarının 8 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 ve daha yakın zamanda profil histonuna aktivitesini karakterize etmek için yaygın bir şekilde kullanılmıştır PTM yazar ve silgi faaliyetleri 24 . /files/ftp_upload/55912/55912fig1.jpg &quot;/&gt; Şekil 1: Bir Histone Peptid mikroarrayinde Antikor Taramasının Kademeli Prosedürünün Çizgi Film Tasvirleri. Tanımlanmış translasyon sonrası modifikasyonları (kırmızı ve mavi daireler) barındıran biyotinlenmiş histon peptidleri, streptavidin kaplı cam üzerinde biyotin-floresein ile birlikte basılmıştır. Pozitif etkileşimler kırmızı floresans olarak görselleştirilir. <a href="http://ecsource.jove.com/files/ftp_upload/55912/55912fig1large.jpg" target="_blank">Bu rakamın daha büyük bir versiyonunu görmek için lütfen tıklayınız.</a>

<table><tbody><tr><td>Printing Buffer</td><td>ArrayIt</td><td>PPB</td><td></td></tr><tr><td>BSA</td><td>Omnipure</td><td>2390</td><td></td></tr><tr><td>Streptavidin-coated glass microscope slides</td><td>Greiner Bio-one</td><td>439003-25</td><td></td></tr><tr><td>polypropylene 384 well plate</td><td>Greiner Bio-one</td><td>784201</td><td></td></tr><tr><td>Biotin-fluorescein</td><td>Sigma</td><td>53608</td><td></td></tr><tr><td>contact microarray printer</td><td>Aushon</td><td>2470</td><td>Aushon 2470 Microarray Printer</td></tr><tr><td>contact microarray printer</td><td>Gene Machines</td><td>OmniGrid 100</td><td>OmniGrid Microarray Printer</td></tr><tr><td>PBS</td><td>Invitrogen</td><td>14190</td><td></td></tr><tr><td>Blocking Buffer</td><td>ArrayIt</td><td>SBB</td><td></td></tr><tr><td>Hydrophobic wax pen</td><td>Vector Labs</td><td>H-4000</td><td>ImmEdge Hydrophobic Barrier PAP Pen</td></tr><tr><td>Silicon Gasket</td><td>Grace Bio-labs</td><td>622511</td><td></td></tr><tr><td>Hybridization Vessel</td><td>Thermo Scientific</td><td>267061</td><td>or similar vessel</td></tr><tr><td>Fluorescent-dye conjugated secondary antibody</td><td>Life Technologies</td><td>A-21244</td><td>Alexa Fluor 647 (anti-rabbit)</td></tr><tr><td>Fluorescent-dye conjugated secondary antibody</td><td>Life Technologies</td><td>A-21235</td><td>Alexa Fluor 647 (anti-mouse)</td></tr><tr><td>Wax Imprinter</td><td>ArrayIt</td><td>MSI48</td><td></td></tr><tr><td>Tween-20</td><td>Omnipure</td><td>9490</td><td></td></tr><tr><td>Microarray Scanner</td><td>Innopsys</td><td>InnoScan 1100AL</td><td>or equivalent microarray scanner</td></tr><tr><td>EipTitan Histone Peptide Microarray</td><td>Epicypher</td><td>112001</td><td></td></tr><tr><td>AbSurance Pro Histone Peptide Microarray</td><td>Millipore</td><td>16668</td><td></td></tr><tr><td>MODified Histone Peptide Array</td><td>Active Motif</td><td>13001</td><td></td></tr><tr><td>Histone Code Peptide Microarrays</td><td>JPT</td><td>His_MA_01</td><td></td></tr><tr><td>Wax</td><td>Royal Oak</td><td>GulfWax</td><td>for wax imprinter</td></tr><tr><td>Humidified Microarray Slide Hybridization Chamber</td><td>VWR</td><td>97000-284</td><td></td></tr><tr><td>High throughput microscope slide washing chamber</td><td>ArrayIt</td><td>HTW</td><td></td></tr><tr><td>Microscope slide centrifuge</td><td>VWR</td><td>93000-204</td><td></td></tr><tr><td>Antibody 1</td><td>Abcam</td><td>8898</td><td></td></tr><tr><td>Antibody 2</td><td>Millipore</td><td>07-473</td><td></td></tr><tr><td>Biotinylated histone peptide</td><td>EpiCypher</td><td>12-0001</td><td>Example peptide. Similar peptides with various modifications are available from several commercial sources.</td></tr><tr><td>ImageMagick</td><td></td><td></td><td>https://www.imagemagick.org/script/index.php</td></tr><tr><td>ArrayNinja</td><td></td><td></td><td>https://rothbartlab.vai.org/tools/</td></tr></tbody></table>

analysis of histone antibody specificity with peptide microarrays

Histon proteinleri üzerindeki translasyon sonrası modifikasyonlar (PTM&#39;ler), kromatin yapısını ve gen ekspresyonunu düzenleme rolleri için geniş çapta incelenmektedir. Histon PTM&#39;lerine spesifik antikorların seri olarak üretilmesi ve dağıtımı, bu işaretler üzerinde araştırmayı büyük ölçüde kolaylaştırdı. Histon PTM antikorları, birçok kromatin biyokimya uygulaması için önemli reaktifler olduğundan, doğru veri yorumlaması ve sahadaki ilerlemenin devam etmesi için antikor özgüllüğünün titiz analizi gereklidir. Bu protokol, histone antikorlarının spesifikliğini profillemek için peptit mikrodizilerinin tasarım, imalat ve kullanımı için entegre bir boru hattı tarif eder. Bu prosedürün tasarım ve analiz yönleri ArrayNinja tarafından kolaylaştırılmış ve açık kaynaklı ve etkileşimli bir yazılım paketi, son zamanlarda mikroarray baskı formatlarının uyarlanmasını hızlandırmak için geliştirdi. Bu boru hattı, piyasada bulunan ve yaygın olarak kullanılan histon PTM antibodisinin çok sayıda taranması için kullanılmıştırS ve bu deneylerden elde edilen veriler çevrimiçi ve genişletici bir Histon Antikor Özgüllilik Veritabanı aracılığıyla serbestçe elde edilebilir. Histonların ötesinde, burada açıklanan genel metod, PTM&#39;ye spesifik antikorların analizine genel olarak uygulanabilir.

Bu protokol, histone PTM antikor özgünlüğünün analizi için bir peptid mikroarray platformu tasarlamak ve üretmek için kullanılmıştır. Çekirdek ve varyant histon proteinleri 38 üzerinde bulunan PTMS bilinen kombinasyonların birçok temsil - (uzunluğu 40 kalıntıları 20) dizi 300&#39;den benzersiz peptid özellikleri bir kütüphane sorgular. Bu boru hattı, yaygın olarak kullanılan ve piyasada mevcut olan histon PTM antikorlarının taranması ...

Watch this Scientific Journal Video about Analysis of Histone Antibody Specificity with Peptide Microarrays at JoVE.com

Analysis of Histone Antibody Specificity with Peptide Microarrays

Histon Antikor Özgüllüğünün Peptit Mikroarraylerle Analizi

Bu el yazısı, histonları ve bunların translasyon sonrası modifikasyonlarını tanıyan antikorların spesifiklik profiline peptid mikroarray teknolojisinin uygulanması için yöntemleri açıklamaktadır.

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

analysis-of-histone-antibody-specificity-with-peptide-microarrays

Research

JoVE Journal

Biochemistry

39.9K Görüntüleme.  Van Andel Research Institute.  Bu el yazısı, histonları ve bunların translasyon sonrası modifikasyonlarını tanıyan antikorların spesifiklik profiline peptid mikroarray teknolojisinin uygulanması için yöntemleri açıklamaktadır. 

Video: Analysis of Histone Antibody Specificity with Peptide Microarrays

Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against human leukocyte antigens (HLA). Histocompatibility guidelines are based on laboratory tests of anti-HLA immunity, which presents either as pre-existing or de novo generated HLA antibodies that constitute a major transplantation barrier. Current tests are built on a single-antigen beads (SAB) platform using a fixed set of ~100 preselected recombinant HLA antigens to probe transplant sera. However, in humans there exist a far greater variety of HLA types, with no two individuals other than identical twins who can share the same combination of HLA sequences. While advanced technologies for HLA typing and direct sequencing can precisely capture any mismatches in DNA sequence between a donor's and recipient's HLA, the SAB assay, due to its limited variety in sequence representation, is unable to precisely detect alloantibodies specifically against the donor HLA mismatches. We sought to develop a complementary method using a different technology to detect and characterize anti-donor HLA antibodies on a personalized basis. The screening tool is a custom peptide array of donor HLA-derived sequences for probing post-transplant sera of the organ recipient to assess the risk for antibody-mediated rejection. On a single array for one donor-recipient pair, up to 600 unique peptides are made based on the donor's HLA protein sequences, each peptide carrying at least one mismatched residue in a 15-amino acid sequence. In our pilot experiments to compare antigen patterns for pre- and post-transplant sera on these arrays, we were able to detect anti-HLA signals with the resolution that also allowed us to pinpoint the immune epitopes involved. These personalized antigen arrays allow high-resolution detection of donor-specific HLA epitopes in organ transplantation.

Mismatches in human leukocyte antigen (HLA) sequences between organ donor and recipient pairs are the major cause of antibody-mediated rejection in organ transplantation. Here we present the use of custom antigen arrays that are based on individual donors' HLA sequences to probe anti-donor HLA alloantibodies in organ recipients.

Peptid diziler HLA Alloantibodies Organ nakli olarak algılanması için kişiselleştirilmiş

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against ...

Biyokimya

Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science of investigating the composition of peptides presented by major histocompatibility complex (MHC) class I and class II molecules using mass spectrometry (MS) technology platforms. Among all the steps in an MS-based immunopeptidomics workflow, sample preparation is critically important for capturing high-quality data of therapeutic relevance. Here, step-by-step instructions are described to isolate MHC class I and II-associated peptides by immunoaffinity purification from quality control samples, from mouse (EL4 and A20), and human (JY) cell lines more specifically. The various reagents and specific antibodies are thoroughly described to isolate MHC-associated peptides from these cell lines, including the steps to verify the beads-binding efficiency of the antibody and the elution efficiency of the MHC-peptide complexes from the beads. The protocol can be used to establish and standardize an immunopeptidomics workflow, as well as to benchmark new protocols. Moreover, the protocol represents a great starting point for any non-experts in addition to foster the intra- and inter-laboratory reproducibility of the sample preparation procedure in immunopeptidomics.

Here, we present a protocol for the purification of MHC class I and class II peptide complexes from mouse and human cell lines providing high-quality immunopeptidomics data. The protocol focuses on sample preparation using commercially available antibodies.

İmmünopeptidomik: Kütle Spektrometresi Analizi için Fare ve İnsan MHC Sınıfı I ve II İlişkili Peptitlerin İzolasyonu

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science ...

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that may facilitate the development of vaccines and peptide drugs. Peptide scanning is a simple and efficient method that straightforwardly maps the linear epitope recognized by a monoclonal antibody (mAb). Here, the authors present an epitope determination methodology involving serially truncated recombinant proteins, synthetic peptide design, and dot-blot hybridization for the antigenic recognition of nervous necrosis virus coat protein using a neutralizing mAb. This technique relies on the dot-blot hybridization of synthetic peptides and mAbs on a polyvinylidene fluoride (PVDF) membrane. The minimum antigenic region of a viral coat protein recognized by the RG-M56 mAb can be narrowed down by step-by-step trimmed peptide mapping onto a 6-mer peptide epitope. In addition, alanine scanning mutagenesis and residue substitution can be performed to characterize the binding significance of each amino acid residue making up the epitope. The residues flanking the epitope site were found to play critical roles in peptide conformation regulation. The identified epitope peptide may be used to form crystals of epitope peptide-antibody complexes for an x-ray diffraction study and functional competition, or for therapeutics.

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

Peptid, bir monoklonal antikor tarafından tanınan lineer B-hücresi epitopunun belirlenmesi: Tarama destekli

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

İmmünoloji ve Enfeksiyon

Zika Virus Specific Diagnostic Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

Zika virüs belirli tanılama Epitope bulma

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

Kompleks Örnekleri spesifik protein Glikozilasyon arasında Multiplexed Yüksek verim Profilleme için Kimyasal-bloke Antikor Mikroarray

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Biyoloji

Analysis of HBV-Specific CD4 T-cell Responses and Identification of HLA-DR-Restricted CD4 T-Cell Epitopes Based on a Peptide Matrix

CD4 T cells play important roles in the pathogenesis of chronic hepatitis B. As a versatile cell population, CD4 T cells have been classified as distinct functional subsets based on the cytokines they secreted: for example, IFN-&#947; for CD4 T helper 1 cells, IL-4 and IL-13 for CD4 T helper 2 cells, IL-21 for CD4 T follicular helper cells, and IL-17 for CD4 T helper 17 cells. Analysis of hepatitis B virus (HBV)-specific CD4 T cells based on cytokine secretion after HBV-derived peptides stimulation could provide information not only about the magnitude of HBV-specific CD4 T-cell response but also about the functional subsets of HBV-specific CD4 T cells. Novel approaches, such as transcriptomics and metabolomics analysis, could provide more detailed functional information about HBV-specific CD4 T cells. These approaches usually require isolation of viable HBV-specific CD4 T cells based on peptide-major histocompatibility complex-II multimers, while currently the information about HBV-specific CD4 T-cell epitopes is limited. Based on an HBV-derived peptide matrix, a method has been developed to evaluate HBV-specific CD4 T-cell responses and identify HBV-specific CD4 T-cell epitopes simultaneously using peripheral blood mononuclear cells samples from chronic HBV infection patients.

Based on a hepatitis B virus (HBV)-derived peptide matrix, HBV-specific CD4 T-cell responses could be evaluated in parallel with identification of HBV-specific CD4 T-cell epitopes.

Peptit Matrisi Bazlı HLA-DR-Restricted CD4 T-Cell Epitoplarının HBV'ye Özgü CD4 T hücre yanıtlarının analizi ve tanımlanması

CD4 T cells play important roles in the pathogenesis of chronic hepatitis B. As a versatile cell population, CD4 T cells have been classified as distinct ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Peptid Epitopların Kantitatif Analiz için bir yüksek veri akışı MHC II Bağlanma Deneyi

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

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 ...

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.

Peptide arrays synthesized by the SPOT method can be used to analyze the substrate specificity of Protein lysine methyltransferases (PKMTs) and to define the substrate spectrum of PKMTs to understand their biological role. This protocol describes how to synthesize peptide arrays, methylate them with PKMTs, and analyze the results.

SPOT Peptit Diziler Protein Lizin Methyltransferases Özgünlüğü Analizi

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays ...

A Peptide Array-Based Detection of Anti-donor HLA Alloantibodies in Organ Recipients

Source: Liu, P., et al. <a href="https://app.jove.com/v/56278">Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation.</a> J. Vis. Exp. (2017).
This video demonstrates an assay to detect anti-donor HLA alloantibodies in organ recipients. A peptide array is synthesized to represent potential epitopes for antibody-mediated organ rejection. The array is incubated with the recipient&#39;s serum, containing alloantibodies that bind to their target epitopes. Antibody binding is detected using chemiluminescence to identify donor-specific HLA epitopes that incite alloantibody reactivity.

Organ Alıcılarında Anti-donör HLA Alloantikorlarının Peptit Dizisi Tabanlı Tespiti

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Design of Custom Array Layout and ...