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

Questo lavoro è stato sostenuto in parte da Van Andel Research Institute e da una borsa di studio degli Istituti Nazionali di Sanità (CA181343) a SBR

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L&#39;affidabilità degli anticorpi nelle applicazioni di ricerca biomedica è fondamentale 46 , 47 . Ciò è particolarmente vero nella biochimica cromatica data la posizione degli anticorpi come strumenti chiave per la maggior parte delle tecniche sviluppate per caratterizzare l&#39;abbondanza e la distribuzione di PTM istoniche. Il protocollo qui presentato illustra una pipeline ottimizzata per la progettazione, la fabbricazione e l&#39;uso di microarray pept...

Il DNA genomico è imballato elegantemente all&#39;interno del nucleo delle cellule eucariotiche con proteine ​​dell&#39;istone per formare la cromatina. La subunità ripetizione della cromatina è il nucleosoma, che consiste di 147 coppie di basi di DNA avvolto attorno ad un nucleo octameric di proteine istoni H2A, - H2B, H3, H4 e 1. La cromatinica è ampiamente organizzata in euchromatin pienamente ricoperti e domini eterocromatina strettamente confezionati. Il grado di compattazione della cromatina regola la misura in cui i macchinari di proteine ​​possono accedere al DNA sottostante per eseguire processi fondamentali del DNA come la replica, la trascrizione e la riparazione. I regolatori chiave dell&#39;accessibilità del genoma nel contesto della cromatina sono PTM sui nodi non strutturati e nuclei delle proteine ​​istone 2 , 3 . Histon PTMs funziona direttamente influenzando la struttura della cromatina 4 e indirettamenteH l&#39;assunzione di proteine ​​di lettura e dei loro complessi macromolecolari associati con attività di ricostruzione cromatica, enzimatica e di ponteggi 5 . Studi sulla funzione istone PTM negli ultimi due decenni suggeriscono in maniera massiccia che questi segni svolgono ruoli chiave nel regolare il destino cellulare, lo sviluppo organico e l&#39;iniziazione / progressione delle malattie. Alimentata dai progressi della tecnologia proteomica basata sulla spettrometria di massa, sono stati scoperti più di 20 PTM istonici unici su più di 80 residui istoniali distinti 6 . Notevolmente, queste modifiche spesso si verificano in combinazioni e coerenti con l&#39;ipotesi del codice istone, numerosi studi suggeriscono che le proteine ​​del lettore sono destinate a regioni discrete di cromatina attraverso il riconoscimento di combinazioni specifiche di istone PTMs 7 , 8 , 9 . Una sfida fondamentale in avanti sarà attribuire funzioni al grGrazie alla lista degli istoni PTMs e per determinare come combinazioni specifiche di PTM istoni orchestrino le funzioni dinamiche associate alla cromatina. Gli anticorpi sono i reagenti lynchpin per la rilevazione di PTM istonico. In quanto tale, più di 1.000 anticorpi specifici PTM-istone sono stati commercialmente sviluppati per l&#39;uso nella ricerca biochimica cromatina. Con il rapido sviluppo di high-throughput tecnologia di sequenziamento del DNA, questi reagenti sono stati ampiamente utilizzati dai singoli ricercatori e epigenomics grandi &quot;calendario&quot; iniziative (ad esempio, codificare e progetto), in ChIP-seq (cromatina immunoprecipitazione accoppiato con sequenziamento di prossima generazione ) Per generare mappe spaziali ad alta risoluzione della distribuzione istone PTM a livello genomico 10 , 11 . Tuttavia, recenti studi hanno dimostrato che la specificità degli anticorpi PTM dell&#39;istone può essere altamente variabile e che questi reagenti presentano<html> Proprietà avorabili come il riconoscimento epitopico fuori bersaglio, forte influenza positiva e negativa da parte dei PTM vicini e difficoltà a discriminare l&#39;ordine di modifica su un determinato residuo ( ad esempio mono-, di- o tri-metililossina) 12 , 13 , 14 , 15 , 16 , 17 , 18 . Pertanto, è necessario un controllo rigoroso della qualità dei reagenti anticorpo specifico PTM-istone per interpretare con precisione i dati generati con questi reagenti preziosi. La tecnologia Microarray consente di interrogare simultaneamente migliaia di interazioni macromolecolari in un formato ad alta produttività, riproducibile e miniaturizzato. Per questo motivo, sono state create diverse piattaforme di microarray per analizzare il DNA proteico 19 ,&quot;&gt; 20, proteine-proteine 21 e proteine-peptide interazioni 22. In effetti, i microarray di peptide dell&#39;istone sono emerse come una piattaforma di scoperta informativa per la ricerca di biochimica cromatica, consentendo una profilazione ad alta velocità degli scrittori, cancellatori e lettori di istone PTMs 15 , 23 , 24 e anche per l&#39;analisi della specificità degli anticorpi degli istoni 17 , 25. Oltre alla loro applicazione nella ricerca di cromatina e epigenetica, gli array di peptone istonico hanno potenziale utilità come test diagnostico / prognostico per il lupus eritematoso sistemico e altre malattie autoimmuni, Gli anticorpi cromatici vengono generati 26 , 27 . Qui descriviamo una pipeline integrata che abbiamo sviluppato per la progettazione, la fabbricazione e la queMicroarrays di peptide istone per generare profili di specificità per gli anticorpi che riconoscono gli istoni e le loro PTM. Il gasdotto è facilitata da ArrayNinja, un open-source, un&#39;applicazione software interattivo che abbiamo recentemente sviluppato, che integra le fasi di progettazione e di analisi di esperimenti di microarray 28. ArrayNinja funziona meglio in Google Chrome. In breve, viene utilizzata una stampante microarray a contatto robotizzato per depositare una biblioteca di peptidi istoni coniugati biotina in posizioni definite su vetrini a microscopio vetrato con streptavidina. Le matrici possono quindi essere utilizzate in un modo conciso e in parallelo di test per interrogare le interazioni con gli anticorpi-epitopi ( Figura 1 ). La biblioteca del peptide è costituita da centinaia di peptidi sintetici unici che ospitano PTM (acetilazione lisina, metilazione lisina / arginina e fosforilazione serina / treonina) da soli e in combinazioni pertinenti in gran parte derivate da set di dati proteomici. Metodi per la sintesi e la convalida dei peptidi<html> Sono dettagliate altrove 23 . I dati generati dagli sforzi di screening degli anticorpi PTH istantanei in corso utilizzando questa piattaforma di array sono archiviati in una risorsa web pubblica, il database di specificità degli anticorpi Histone (www.histoneantibodies.com). In particolare, i microarray di peptide istone fabbricati con variazioni di questo protocollo sono stati ampiamente utilizzati per caratterizzare l&#39;attività dei domini di lettore di istone PTM 8 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 e più recentemente all&#39;istone del profilo PTM scrittore e attività gomma 24 . /files/ftp_upload/55912/55912fig1.jpg &quot;/&gt; Figura 1: Disposizione del fumetto della procedura stepwise per la screening degli anticorpi su un microarray di peptide dell&#39;istone. I peptidi biotinilati di istone che ospitano modifiche post-traslazionali definite (cerchi rossi e blu) sono stampati con biotina-fluoresceina sul vetro rivestito con streptavidina. Le interazioni positive sono visualizzate come fluorescenza rossa. <a href="http://ecsource.jove.com/files/ftp_upload/55912/55912fig1large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</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

Le modificazioni post-traduzionali (PTMs) sulle proteine ​​dell&#39;istone sono ampiamente studiate per i loro ruoli nella regolazione della struttura cromatica e dell&#39;espressione genica. La produzione di massa e la distribuzione di anticorpi specifici ai PTM istonici ha facilitato notevolmente la ricerca su questi segni. Poiché gli anticorpi PTM dell&#39;istone sono reagenti chiave per molte applicazioni di biochimica cromatica, è necessaria un&#39;analisi rigorosa della specificità degli anticorpi per un&#39;accurata interpretazione dei dati e un progresso continuo nel campo. Questo protocollo descrive una pipeline integrata per la progettazione, la fabbricazione e l&#39;uso di microarray peptidi per la profilazione della specificità degli anticorpi istoni. Gli aspetti di progettazione e analisi di questa procedura sono facilitati da ArrayNinja, un pacchetto software open source e interattivo che abbiamo recentemente sviluppato per semplificare la personalizzazione dei formati di stampa con microarray. Questa pipeline è stata utilizzata per la visualizzazione di un gran numero di anticorpi anti-PTM di istone commercialmente disponibili e ampiamente utilizzatiE i dati generati da questi esperimenti sono liberamente disponibili tramite un database di specificità anticorpo istante in linea e in espansione. Al di là degli istoni, la metodologia generale descritta qui può essere applicata ampiamente all&#39;analisi degli anticorpi PTM-specifici.

Questo protocollo è stato utilizzato per progettare e realizzare una piattaforma microarray peptide per l&#39;analisi della specificità degli anticorpi PTM. L&#39;array richiede una libreria di più di 300 funzionalità peptide uniche (20-40 residui di lunghezza) che rappresentano molte delle combinazioni note di PTM trovate sulle proteine ​​di istone del nucleo e delle varianti 38 . Questa pipeline è stata un cavallo di lavoro per lo screening di molti ant...

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

Analisi della specificità degli anticorpi istonici con microarray di peptide

Questo manoscritto descrive i metodi per applicare la tecnologia del microarray peptide alla profilazione di specificità degli anticorpi che riconoscono gli istoni e le loro modificazioni post-traslazionali.

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 Views.  Van Andel Research Institute.  Questo manoscritto descrive i metodi per applicare la tecnologia del microarray peptide alla profilazione di specificità degli anticorpi che riconoscono gli istoni e le loro modificazioni post-traslazionali. 

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.

Personalizzato Peptide matrici per il rilevamento di anticorpi HLA nei trapianti d'organo

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

Ricerca

Biochimica

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.

Immunopeptidomics: Isolamento di peptidi associati A MHC di classe I e II di topi e umani per l'analisi della spettrometria di massa

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.

Identificazione di un Epitopo Lineare B-cell monoclonale riconosciuto Peptide scansione-assistito

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

Immunologia e infezioni

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.

Virus di Zika specifico diagnostico dell'epitopo 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 ...

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.

Anticorpo Microarray chimicamente bloccato per Multiplexed high-throughput Profiling di glicosilazione delle proteine ​​specifico in campioni complessi

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

Biologia

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.

Analisi delle risposte delle cellule T CD4 specifiche dell'HBV e identificazione degli epitopi delle cellule T CD4 con restrizioni HLA-DR basati su una matrice peptidica

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.

Un saggio di legame High Throughput MHC II per l&#39;analisi quantitativa dei peptidi epitopi

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.

Identificare proteina-proteina siti di interazione Uso Peptide Array

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.

Analisi Specificità di proteine ​​lisina metiltransferasi Utilizzando SPOT Peptide Array

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.

Un rilevamento basato su array di peptidi di alloanticorpi HLA anti-donatore nei riceventi di organi

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

Analisi della specificità degli anticorpi istonici con microarray di peptide

Riepilogo

Esplora Altri Video

Capitoli in questo video

Personalizzato Peptide matrici per il rilevamento di anticorpi HLA nei trapianti d'organo

Immunopeptidomics: Isolamento di peptidi associati A MHC di classe I e II di topi e umani per l'analisi della spettrometria di massa

Identificazione di un Epitopo Lineare B-cell monoclonale riconosciuto Peptide scansione-assistito

Virus di Zika specifico diagnostico dell'epitopo Discovery

Anticorpo Microarray chimicamente bloccato per Multiplexed high-throughput Profiling di glicosilazione delle proteine specifico in campioni complessi

Analisi delle risposte delle cellule T CD4 specifiche dell'HBV e identificazione degli epitopi delle cellule T CD4 con restrizioni HLA-DR basati su una matrice peptidica

Un saggio di legame High Throughput MHC II per l'analisi quantitativa dei peptidi epitopi

Identificare proteina-proteina siti di interazione Uso Peptide Array

Analisi Specificità di proteine lisina metiltransferasi Utilizzando SPOT Peptide Array

Un rilevamento basato su array di peptidi di alloanticorpi HLA anti-donatore nei riceventi di organi

Analisi della specificità degli anticorpi istonici con microarray di peptide

Riepilogo

Esplora Altri Video

Capitoli in questo video

Personalizzato Peptide matrici per il rilevamento di anticorpi HLA nei trapianti d'organo

Immunopeptidomics: Isolamento di peptidi associati A MHC di classe I e II di topi e umani per l'analisi della spettrometria di massa

Identificazione di un Epitopo Lineare B-cell monoclonale riconosciuto Peptide scansione-assistito

Virus di Zika specifico diagnostico dell'epitopo Discovery

Anticorpo Microarray chimicamente bloccato per Multiplexed high-throughput Profiling di glicosilazione delle proteine ​​specifico in campioni complessi

Analisi delle risposte delle cellule T CD4 specifiche dell'HBV e identificazione degli epitopi delle cellule T CD4 con restrizioni HLA-DR basati su una matrice peptidica

Un saggio di legame High Throughput MHC II per l'analisi quantitativa dei peptidi epitopi

Identificare proteina-proteina siti di interazione Uso Peptide Array

Analisi Specificità di proteine ​​lisina metiltransferasi Utilizzando SPOT Peptide Array

Un rilevamento basato su array di peptidi di alloanticorpi HLA anti-donatore nei riceventi di organi

Anticorpo Microarray chimicamente bloccato per Multiplexed high-throughput Profiling di glicosilazione delle proteine specifico in campioni complessi

Analisi Specificità di proteine lisina metiltransferasi Utilizzando SPOT Peptide Array