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

이 연구는 반 안델 연구소 (Van Andel Research Institute)와 국립 보건원 (CA181343)에서 SBR

저자는 공개 할 것이 없습니다.

생물 의학 연구 분야의 항체 신뢰도가 가장 중요합니다 46 , 47 . 이것은 히스톤 PTM의 풍부성과 분포를 특성화하기 위해 개발 된 대부분의 기술에 대한 핵심 도구로서 항체의 위치를 ​​고려할 때 염색질 생화학에서 특히 그렇습니다. 여기에 제시된 프로토콜은 히스톤 PTM 항체 특이성을 분석하기위한 펩타이드 마이크로 어레이의 설계, 제조 및 사용을?...

게놈 DNA는 히스톤 단백질이있는 진핵 세포 핵 내부에 우아하게 포장되어 염색질을 형성합니다. 염색질의 반복적 인 아 단위는 히스톤 단백질 (H2A, H2B, H3 및 H4 1) 의 8 량체 핵 주위에 감싸 진 147 염기쌍의 DNA로 구성된 뉴 소솜 (nucleosome)이다. 크로마 틴은 느슨하게 패킹 된 진균 염색체와 단단히 채워진 헤테로 크로마 틴 도메인으로 광범위하게 구성됩니다. 염색질 압축 정도는 단백질 기계가 복제, 전사 및 수리와 같은 기본적인 DNA 템플릿 공정을 수행하기 위해 기본 DNA에 접근 할 수있는 정도를 조절합니다. 염색체의 맥락에서 게놈 접근성의 주요 조절 인자는 히스톤 단백질 2 , 3 의 구조화되지 않은 꼬리와 핵심 부위의 PTM입니다. 히스톤 PTM은 염색질 4 의 구조에 직접적으로 영향을 미치고 간접적으로 기능합니다.염색질 개질, 효소 및 스캐 폴딩 활동을하는 독자 단백질 및 관련 거대 분자 복합체의 모집 5 . 지난 20 년간 히스톤 PTM 기능에 대한 연구는 세포의 운명, 유기체 발달 및 질병의 시작 / 진행을 조절하는 데 중요한 역할을한다는 것을 압도적으로 제안합니다. 질량 분석 기반 단백체 기술의 발전에 힘 입어, 80 명 이상의 서로 다른 히스톤 잔류 물에 20 개 이상의 고유 한 히스톤 PTMS는 6 발견되었다. 주목할 만하게,이 수정은 수시로 조합에서 생기고, &quot;히스톤 부호&quot;가설과 일치하여, 수많은 학문은 독자 단백질이 히스톤 PTMs의 특정한 조합의 승인을 통해 염색질의 불연속 지역에 표적으로 한 ㄴ다는 것을 건의한다 7 , 8 , 9 . 앞으로 나아갈 핵심 과제는 gr에 함수를 할당하는 것입니다.히스톤 PTM의 목록을 작성하고 히스톤 PTM의 특정 조합이 염색질과 관련된 동적 기능을 조율하는 방법을 결정할 수 있습니다. 항체는 히스톤 PTM의 검출을위한 lynchpin 시약입니다. 이와 같이, 염색질 생화학 연구에 사용하기 위해 1,000 개 이상의 히스톤 PTM 특이 적 항체가 상업적으로 개발되었습니다. 고효율 DNA 시퀀싱 기술의 급속한 발전으로이 시약은 ChIP-seq (염색질 면역 침전 및 차세대 시퀀싱)에서 개별 조사자와 대규모 후성 유전체학 &quot;로드맵&quot;이니셔티브 ( 예 : ENCODE 및 BLUEPRINT)에 광범위하게 사용되고 있습니다 ) 파이프 라인은 히스톤 PTM 분포 게놈 전체의 고해상도 공간 맵을 생성합니다 10 , 11 . 그러나 최근의 연구에 따르면 히스톤 PTM 항체의 특이성은 매우 다양 할 수 있으며 이러한 시약은 unfones<html> 오프 - 타겟 에피토프 인식, 이웃 PTM에 의한 강한 긍정 및 부정적인 영향, 특정 잔기 ( 예 : 모노 -, 디 - 또는 트리 - 메틸 리신)의 변형 순서 식별 어려움과 같은 유용한 특성 12 , 13 , 14 , 15 , 16 , 17 , 18 . 따라서 히스톤 PTM 특이 적 항체 시약의 엄격한 품질 관리가 이러한 유용한 시약으로 생성 된 데이터를 정확하게 해석하는 데 필요합니다. Microarray 기술은 높은 처리량, 재현성 및 소형화 된 포맷으로 수천 개의 거대 분자 상호 작용을 동시에 조사 할 수있게합니다. 이러한 이유로, 단백질 -DNA 19 을 분석하기 위해 다양한 마이크로 어레이 플랫폼이 만들어졌으며 ,&quot;&gt; (20), 단백질 - 단백질 21 단백질 펩타이드의 상호 작용 (22). 실제로, 히스톤 펩타이드 마이크로 어레이는 작가, 지우개의 높은 처리량 프로파일 링을 가능하게 염색질 생화학 연구에 대한 정보를 검색 플랫폼으로 등장, 그리고 히스톤의 독자 PTMS 15있다 , 23, 24, 또한 히스톤 항체 특이성의 분석을 위해 17, 25. 염색질과 후성 유전학 연구에 자신의 응용 프로그램을 넘어, 히스톤 펩타이드 배열은 전신성 홍 반성 루푸스 등의자가 면역 질환에 대한 진단 / 예후 테스트 잠재적 인 유틸리티가 어디 방지 염색질자가 항체가 생성됩니다 26 , 27 . 여기서는 설계, 제작 및 큐를 위해 개발 한 통합 파이프 라인을 설명합니다.히스톤 및 그 PTM을 인식하는 항체에 대한 특이성 프로파일을 생성하기 위해 히스톤 펩타이드 마이크로 어레이를 제조한다. 파이프 라인은 ArrayNinja, 마이크로 어레이 실험 (28)의 설계 및 분석 단계를 통합하는 오픈 소스, 우리는 최근에 개발 된 대화 형 소프트웨어 응용 프로그램에 의해 촉진된다. ArrayNinja는 Google 크롬에서 가장 잘 작동합니다. 간단히 말하면, 로봇 접촉 마이크로 어레이 프린터를 사용하여 스트렙 타비 딘 - 코팅 된 유리 현미경 슬라이드상의 한정된 위치에 비오틴 - 컨쥬 게이트 된 히스톤 펩타이드의 라이브러리를 침착시킨다. 그런 다음 항체 - 항원 결정기 상호 작용을 조사하기 위해 경쟁적이고 평행 한 분석 형식으로 배열을 사용할 수 있습니다 ( 그림 1 ). 펩타이드 라이브러리는 PTM (라이신 아세틸 화, 라이신 / 아르기닌 메틸화, 및 세린 / 트레오닌 인산화)이있는 수백 가지의 독특한 합성 펩타이드와 프로테오믹스 데이터 세트에서 주로 유래 된 적절한 조합으로 구성됩니다. 펩타이드 합성 및 검증 방법<html> 다른 곳에서 (23)에 자세히 설명되어 있습니다. 이 어레이 플랫폼을 사용하는 진행중인 히스톤 PTM 항체 스크리닝 작업에서 생성 된 데이터는 공개 웹 리소스 인 Histone Antibody Specificity Database (www.histoneantibodies.com)에 보관됩니다. 특히,이 프로토콜의 변형으로 제작 된 히스톤 펩타이드 마이크로 어레이는 히스톤 PTM 판독기 도메인 8 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 의 활성을 특성화하기 위해 광범위하게 사용되어왔다. PTM 작성자 및 지우개 활동 24 . /files/ftp_upload/55912/55912fig1.jpg &quot;/&gt; 그림 1 : 히스톤 펩타이드 마이크로 어레이에서 항체 스크리닝을위한 Stepwise 절차의 카툰 묘사. 정의 된 번역 후 변형 (적색 및 청색 원)을 갖는 비 오티 닐화 히스톤 펩타이드는 스트렙 타비 딘 - 코팅 된 유리상에 바이오틴 - 플루오 레세 인과 함께 인쇄된다. 양성 상호 작용은 적색 형광으로 시각화됩니다. <a href="http://ecsource.jove.com/files/ftp_upload/55912/55912fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</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

히스톤 단백질에 대한 번역 후 변형 (PTMs)은 염색질 구조 및 유전자 발현 조절에서의 역할에 대해 널리 연구되고있다. 히스톤 PTM에 특이적인 항체의 대량 생산 및 분포는 이러한 표지에 대한 연구를 크게 촉진시켰다. 히스톤 PTM 항체는 많은 염색질 생화학 분야의 주요 시약이기 때문에 항체 특이성의 엄격한 분석이 정확한 데이터 해석과 현장에서의 지속적인 진보에 필요합니다. 이 프로토콜은 히스톤 항체의 특이성을 프로파일 링하기위한 펩타이드 마이크로 어레이의 설계, 제조 및 사용을위한 통합 파이프 라인을 설명합니다. 이 절차의 설계 및 분석 측면은 Microarray 인쇄 형식의 사용자 정의를 간소화하기 위해 최근 개발 한 오픈 소스 및 대화 형 소프트웨어 패키지 인 ArrayNinja를 사용하여 쉽게 수행 할 수 있습니다. 이 파이프 라인은 상업적으로 이용되고 널리 사용되고있는 히스톤 PTM 항체이 실험에서 생성 된 데이터는 온라인 및 확장 Histone Antibody Specificity Database를 통해 자유롭게 사용할 수 있습니다. 히스톤을 넘어, 여기에 기술 된 일반적인 방법론은 PTM 특이 적 항체의 분석에 광범위하게 적용될 수 있습니다.

이 프로토콜은 히스톤 PTM 항체 특이성의 분석을위한 펩타이드 마이크로 어레이 플랫폼을 설계하고 제조하는데 사용되었습니다. 이 어레이는 핵심 및 변형 히스톤 단백질에서 발견되는 PTM의 많은 조합을 나타내는 300 개 이상의 고유 한 펩타이드 기능 (20-40 잔기 길이) 라이브러리를 조회합니다 38 . 이 파이프 라인은 널리 사용되고 상업적으로 이용 가능?...

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

펩티드 Microarrays를 가진 Histone 항체 특이성의 분석

이 원고는 히스톤 및 그 번역 후 변형을 인식하는 항체의 특이성 프로파일 링에 펩타이드 마이크로 어레이 기술을 적용하는 방법을 설명합니다.

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 조회수.  Van Andel Research Institute.  이 원고는 히스톤 및 그 번역 후 변형을 인식하는 항체의 특이성 프로파일 링에 펩타이드 마이크로 어레이 기술을 적용하는 방법을 설명합니다. 

비디오: 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.

장기 이식에서 HLA Alloantibodies의 검출에 대 한 펩 티 드 배열 맞춤

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

연구

생화학

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.

면역 펩티도믹스: 질량 분광분석 분석을 위한 마우스 및 인간 MHC 급 I-및 II 관련 펩타이드의 격리

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.

펩타이드 모노클로 날 항체 인식 선형 B 세포 에피토프의 확인을 스캔 보조

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

면역학 및 감염병학

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 바이러스 특정 진단 Epitope 발견

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.

단지 샘플의 특정 단백질 글리코 실화의 다중 높은 처리량 프로파일을위한 화학을 차단 항체 Microarray

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

생물학

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.

펩타이드 매트릭스에 근거한 HLA-DR-제한 CD4 T 세포 에피토프의 HBV 특정 CD4 T 세포 반응 및 식별의 분석

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.

펩티드 에피토프의 정량 분석​​을위한 높은 처리량 MHC II 결합 분석

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.

펩타이드 배열을 사용하여 단백질 - 단백질 상호 작용 사이트를 확인

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 펩타이드 배열을 사용하여 단백질 리신 Methyltransferases의 특이성 분석

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.

장기 수혜자에서 항공여자 HLA 동종항체의 펩타이드 어레이 기반 검출

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

펩티드 Microarrays를 가진 Histone 항체 특이성의 분석

요약

더 많은 비디오 탐색

이 비디오의 챕터

장기 이식에서 HLA Alloantibodies의 검출에 대 한 펩 티 드 배열 맞춤

면역 펩티도믹스: 질량 분광분석 분석을 위한 마우스 및 인간 MHC 급 I-및 II 관련 펩타이드의 격리

펩타이드 모노클로 날 항체 인식 선형 B 세포 에피토프의 확인을 스캔 보조

Zika 바이러스 특정 진단 Epitope 발견

단지 샘플의 특정 단백질 글리코 실화의 다중 높은 처리량 프로파일을위한 화학을 차단 항체 Microarray

펩타이드 매트릭스에 근거한 HLA-DR-제한 CD4 T 세포 에피토프의 HBV 특정 CD4 T 세포 반응 및 식별의 분석

펩티드 에피토프의 정량 분석을위한 높은 처리량 MHC II 결합 분석

펩타이드 배열을 사용하여 단백질 - 단백질 상호 작용 사이트를 확인

SPOT 펩타이드 배열을 사용하여 단백질 리신 Methyltransferases의 특이성 분석

장기 수혜자에서 항공여자 HLA 동종항체의 펩타이드 어레이 기반 검출

펩티드 Microarrays를 가진 Histone 항체 특이성의 분석

요약

더 많은 비디오 탐색

이 비디오의 챕터

장기 이식에서 HLA Alloantibodies의 검출에 대 한 펩 티 드 배열 맞춤

면역 펩티도믹스: 질량 분광분석 분석을 위한 마우스 및 인간 MHC 급 I-및 II 관련 펩타이드의 격리

펩타이드 모노클로 날 항체 인식 선형 B 세포 에피토프의 확인을 스캔 보조

Zika 바이러스 특정 진단 Epitope 발견

단지 샘플의 특정 단백질 글리코 실화의 다중 높은 처리량 프로파일을위한 화학을 차단 항체 Microarray

펩타이드 매트릭스에 근거한 HLA-DR-제한 CD4 T 세포 에피토프의 HBV 특정 CD4 T 세포 반응 및 식별의 분석

펩티드 에피토프의 정량 분석​​을위한 높은 처리량 MHC II 결합 분석

펩타이드 배열을 사용하여 단백질 - 단백질 상호 작용 사이트를 확인

SPOT 펩타이드 배열을 사용하여 단백질 리신 Methyltransferases의 특이성 분석

장기 수혜자에서 항공여자 HLA 동종항체의 펩타이드 어레이 기반 검출

펩티드 에피토프의 정량 분석을위한 높은 처리량 MHC II 결합 분석