この研究は、部分的には、Van Andel Research Instituteによって支援され、国立衛生研究所（CA181343）からSBR

著者は何も開示することはない。

生物医学研究用途における抗体の信頼性が最優先事項46、47です。これは、ヒストンPTMの存在量および分布を特徴付けるために開発された技術の大部分のための重要なツールとしての抗体の位置を考慮すると、クロマチン生化学において特に当てはまります。ここに示されたプロトコルは、ヒストンPTM抗体特異性を分析するためのペプチドマイクロ?...

ゲノムDNAは、ヒストンタンパク質を含む真核細胞の核内にエレガントにパッケージングされ、クロマチンを形成する。クロマチンの反復サブユニットはヌクレオソームであり、これはヒストンタンパク質の八量体コア（H2A、H2B、H3、およびH4 1）のまわりに包まれた147塩基対のDNAからなる。クロマチンは、ゆるやかに詰まったユークロマチンおよび密に詰まったヘテロクロマチンドメインに広く組織されている。クロマチン圧縮の程度は、複製、転写、および修復のような基本的なDNA鋳型プロセスを実行するために、タンパク質機械が根底にあるDNAにアクセスできる程度を調節する。 クロマチンの文脈におけるゲノムアクセシビリティの重要な調節因子は、ヒストンタンパク質2、3の構造化されていない尾とコアドメイン上のPTMです。ヒストンPTMは、クロマチン4の構造に直接的に作用し、間接的に作用する染色体リモデリング、酵素的および足場活性を有するリーダータンパク質および関連する巨大分子複合体の動員5 。過去20年にわたるヒストンPTM機能の研究は、これらのマークが細胞運命、生物発達、および疾患の開始/進行の調節において重要な役割を果たすことを圧倒的に示唆している。質量分析に基づくプロテオーム技術の進歩により、80以上の異なるヒストン残基に20以上のユニークなヒストンPTMが発見されました6 。注目すべきことに、これらの修飾は、しばしば組み合わせで発生し、「ヒストンコード」仮説と一致して、多くの研究は、リーダタンパク質がヒストンのPTM 7、8、9の特定の組み合わせの認識を介してクロマチンの別個の領域を標的とすることを示唆しています。重要な課題は、関数をgrに割り当てることですヒストンPTMの特定の組み合わせがどのようにしてクロマチンに関連する動的機能を調整するかを決定することができる。 抗体は、ヒストンPTMの検出のためのリンチピン試薬である。このように、クロマチン生化学研究に使用するために、1,000を超えるヒストンPTM特異的抗体が商業的に開発されている。ハイスループットDNA配列決定技術の急速な発展に伴い、これらの試薬は、チップ配列の個々の研究者および大規模エピゲノミクス「ロードマップ」の取り組み（ 例えば 、ENCODEおよび青写真）によって広く使用されている（次世代シークエンシングと結合クロマチン免疫沈降）パイプラインヒストンPTM分布ゲノムワイド10、11の高解像度空間マップを生成します。しかし、最近の研究により、ヒストンPTM抗体の特異性は非常に可変であり得、これらの試薬は非<html>特定の残基に変更順序を識別ようなオフターゲットエピトープ認識、隣接のPTMによる強い正および負の影響、及び困難としてavorable特性（ 例えば 、モノ- 、ジ- 、またはトリ-メチルリジン）12、13、14、15 、16、17、18。したがって、これらの貴重な試薬で生成されたデータを正確に解釈するには、ヒストンPTM特異的抗体試薬の厳密な品質管理が必要です。 マイクロアレイ技術は、ハイスループットで、再現性があり、かつ小型化されたフォーマットで、何千もの高分子相互作用の同時検査を可能にする。このため、マイクロアレイプラットフォームの様々なタンパク質-DNA 19を分析するために作成されています、「&gt; 20、タンパク質-タンパク質21、及びタンパク質-ペプチド相互作用22。実際、ヒストンペプチドマイクロアレイは消しゴム、作家のハイスループットプロファイリングを可能にする、クロマチン生化学研究のための有益な発見プラットフォームとして浮上し、そしてヒストンのPTM 15の読者ました、23、24、またヒストン抗体特異性17、25の分析のため。クロマチンとエピジェネティクス研究におけるそれらの用途を超えて、ヒストンペプチド配列は、抗全身性エリテマトーデスおよび他の自己免疫疾患の診断/予後診断検査としての潜在的有用性を有しますクロマチン自己抗体は26、27を生成しています。 ここでは、設計、製造、および開発のために開発した統合パイプラインについて説明しますヒストンおよびそれらのPTMを認識する抗体の特異性プロファイルを生成するためにヒストンペプチドマイクロアレイをライニングする。このパイプラインは、我々が最近開発したオープンソースのインタラクティブなソフトウェアアプリケーションであるArrayNinjaによって容易にされ、マイクロアレイ実験の設計と解析段階を統合している28 。 ArrayNinjaはGoogle Chromeで最適に動作します。簡単に述べると、ロボット接触マイクロアレイプリンターを用いて、ストレプトアビジン被覆ガラス顕微鏡スライド上の規定された位置にビオチン結合ヒストンペプチドのライブラリーを沈着させる。次いで、抗体 - エピトープ相互作用を調べるために、競合的および並行アッセイ形式でアレイを使用することができる（ 図1 ）。ペプチドライブラリーは、PTM（リジンアセチル化、リシン/アルギニンメチル化、およびセリン/スレオニンリン酸化）を単独で、プロテオミクスデータセットに主に由来する関連する組み合わせを有する何百ものユニークな合成ペプチドからなる。ペプチド合成および検証のための方法<html>他の場所で詳述されている23 。このアレイプラットフォームを利用した、現在行われているヒストンPTM抗体スクリーニングの取り組みから得られたデータは、公開されているWebリソース、Histone Antibody Specificity Database（www.histoneantibodies.com）に保存されています。注目すべきことに、このプロトコルのバリエーションで製造ヒストンペプチドマイクロアレイはまた、ヒストンPTMリーダードメイン8、29、30、31、32、33、34、35、36、37の活性を特徴づけるために広く使用されており、より最近ヒストンをプロファイルしますPTMライターと消しゴムの活動24 。 /files/ftp_upload/55912/55912fig1.jpg &quot;/&gt; 図1：ヒストンペプチドマイクロアレイ上の抗体スクリーニングのための段階的手順の漫画の描写。定義された翻訳後修飾（赤色および青色の円）を有するビオチン化ヒストンペプチドを、ストレプトアビジン被覆ガラス上のビオチン - フルオレセインと同時プリントする。陽性の相互作用は、赤色蛍光として視覚化される。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55912/55912fig1large.jpg" target="_blank">この図の拡大版を見るには、ここをクリックしてください。</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

ヒストンタンパク質の翻訳後修飾（PTM）は、クロマチン構造および遺伝子発現の調節におけるそれらの役割について広く研究されている。ヒストンPTMに特異的な抗体の大量生産および分布は、これらのマークの研究を非常に容易にした。ヒストンPTM抗体は多くのクロマチン生化学アプリケーションにとって重要な試薬であるため、正確なデータ解釈と現場での継続的な進歩には抗体特異性の厳密な解析が必要です。このプロトコルは、ヒストン抗体の特異性をプロファイリングするためのペプチドマイクロアレイの設計、製作および使用のための統合されたパイプラインを記載している。この手順の設計と分析の側面は、マイクロアレイ印刷フォーマットのカスタマイズを合理化するために最近開発したオープンソースのインタラクティブなソフトウェアパッケージであるArrayNinjaによって容易になります。このパイプラインは、市販されて広く使用されている多数のヒストンPTM抗体のスクリーニングに使用されているこれらの実験から生成されたデータは、オンラインおよび拡大Histone Antibody Specificity Databaseを通じて自由に入手できます。ヒストンを越えて、本明細書に記載の一般的な方法論は、PTM特異的抗体の分析に広く適用することができる。

このプロトコルは、ヒストンPTM抗体特異性の分析のためのペプチドマイクロアレイプラットフォームを設計し、製造するために使用されてきた。このアレイは、コアおよびバリアントヒストンタンパク質38に見られるPTMの既知の組み合わせの多くを表す、300を超える独自のペプチド機能（長さ20〜40残基）のライブラリを照会します38</sup...

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

ペプチドマイクロアレイによるヒストン抗体特異性の解析

この原稿では、ヒストンとその翻訳後修飾を認識する抗体の特異性プロファイリングにペプチドマイクロアレイ技術を適用する方法が記載されています。

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

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

39.8K ビュー.  Van Andel Research Institute. この原稿では、ヒストンとその翻訳後修飾を認識する抗体の特異性プロファイリングにペプチドマイクロアレイ技術を適用する方法が記載されています。 

ビデオ: 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.

臓器移植における HLA 同種抗体の検出のためのペプチド配列をお好み

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

生化学

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.

免疫ペプチドミクス:マウスとヒトMHCクラスI-およびII関連ペプチドの分離による質量分析

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.

モノクローナル抗体認識リニアB細胞エピトープのペプチドスキャンアシスト識別

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

免疫・感染学

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.

ジカ特定診断エピトープの探索

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.

複雑なサンプル中の特定のタンパク質のグリコシル化の多重化ハイスループットプロファイリングのための化学的にブロックされた抗体マイクロアレイ

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

生物学

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.

ペプチドマトリックスに基づくHBV特異的CD4 T細胞応答の解析とHLA-DR制限CD4 T細胞エピトープの同定

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.

ペプチドエピトープの定量分析のための高スループット、MHC II結合アッセイ

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.

ペプチド配列の使用タンパク質間相互作用部位を同定

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ペプチド配列を用いてタンパク質リジンメチルトランスフェラーゼの特異性の解析

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.bdigitaluss.remotexs.co/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.

臓器レシピエントにおける抗ドナーHLA同種抗体のペプチドアレイベースの検出

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