Name: chemically-blocked antibody microarray for multiplexed high-throughput profiling of specific protein glycosylation in complex samples
Uploaded: 2012-05-04T13:00:00
Description: Watch this Scientific Journal Video about Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples at JoVE.com

이 작품은 간염과 바이러스 연구소에 의해 지원되었다.

1. 분석을위한 항체 Microarray 인쇄 <ol><li> 0.5 MG / 인산염 완충 식염수, 산도 7.2 (PBS)의 ML에 모든 항체를 희석. </li><li> 384 잘 소스 판으로 나누어지는 각 항체의 40 μl합니다. </li><li> Scienion의 sciFLEXARRAYER의 microarrayer에 384 잘 소스 판을로드합니다. </li><li> 목표로 microarrayer에 20의 PATH 환경 microarray 슬라이드를로드합니다. </li><li> 27 항체 및 제어 단백질이 9x9 패턴 (그림 1E, 1F)에서 세중의에서...

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1. 대상 단백질과 캡처 항체 선택 항체 microarray 분석에 앞서, 몇 가지 시약 및 재료는 고려가 필요하고 준비가되어 있습니다. glycan 프로 파일링 또는 glycan의 biomarker 검사, 당단백질 후보에 대한 특정 항체의 패널에 대한 항체 microarray를 디자인하기 위해서는 문학에 또는 이전 결과에 따라 결정되어야한다. 이러한 항체는 일반적으로 이러한 R &amp; D 시스템 등 IgGs 등 다양한 업체?...

<table border="1"><tbody><tr><td> 신분증 </td><td> 시약의 이름 </td><td> 약어 </td><td> 회사 </td><td> # 카탈로그 </td></tr><tr><td> L1 </td><td> Biotinylated Concanavalin </td><td> ConA </td><td> 벡터 연구소 </td><td> BK-1000 </td></tr><tr><td> L2 </td><td> Biotinylated Sambucus Nigra의 렉틴 </td><td> SNA </td><td> 벡터 연구소 </td><td> B-1305 </td></tr><tr><td> L3 </td><td> Biotinylated 렌즈 Culinaris Agglutinin </td><td> LCA </td><td> 벡터 연구소 </td><td> BK-2000 </td></tr><tr><td> L4 </td><td> Ricinus Communis Agglutinin 봐도 Biotinylated </td><td> RCA </td><td> 벡터 연구소 </td><td> BK-1000 </td></tr><tr><td> 5 번 </td><td> Biotinylated Aleuria Aurantia의 렉틴 </td><td> AAL </td><td> 벡터 연구소 </td><td> B-1395 </td></tr><tr><td> L6 </td><td> Biotinylated Erythrina Cristagalli의 렉틴 </td><td> ECL </td><td> 벡터 연구소 </td><td> BK-3000 </td></tr><tr><td> L7 </td><td> Biotinylated Griffonia (Bandeiraea) Simplicifolia 렉틴 II </td><td> GSL II </td><td> 벡터 연구소 </td><td> BK-3000 </td></tr><tr><td> L8 </td><td> Biotinylated 밀 배아 Agglutinin </td><td> WGA </td><td> 벡터 연구소 </td><td> BK-1000 </td></tr><tr><td> L9 </td><td> Biotinylated Phaseolus vulgaris의 Erythroagglutinin </td><td> PHA-E </td><td> 벡터 연구소 </td><td> BK-2000 </td></tr><tr><td> 10 파운드 라니 </td><td> Biotinylated Phaseolus vulgaris의 Leucoagglutinin </td><td> PHA-L </td><td> 벡터 연구소 </td><td> BK-2000 </td></tr><tr><td> L11 </td><td> Biotinylated 땅콩 Agglutinin </td><td> PNA </td><td> 벡터 연구소 </td><td> BK-1000 </td></tr><tr><td> L12 </td><td> Pisum Sativum Agglutinin을 Biotinylated </td><td> PSA </td><td> 벡터 연구소 </td><td> BK-2000 </td></tr><tr><td> L13 </td><td> Biotinylated Dolichos Biflorus Agglutinin </td><td> DBA </td><td> 벡터 연구소 </td><td> BK-1000 </td></tr><tr><td> L14 </td><td> Biotinylated 흰독말풀의 가시독말풀의 렉틴 </td><td> DSL </td><td> 벡터 연구소 </td><td> BK-3000 </td></tr><tr><td> L15 </td><td> Biotinylated Sophora Japonica Agglutinin </td><td> SJA </td><td> 벡터 연구소 </td><td> BK-2000 </td></tr><tr><td> L16 </td><td> Biotinylated 대두 Agglutinin </td><td> SBA </td><td> 벡터 연구소 </td><td> BK-1000 </td></tr><tr><td> L17 </td><td> Biotinylated Solanum Tuberosum (감자) 렉틴 </td><td> STL </td><td> 벡터 연구소 </td><td> BK-3000 </td></tr><tr><td> L18 </td><td> Biotinylated Griffonia (밴드eiraea) Simplicifolia의 렉틴 I </td><td> GSL 전 </td><td> 벡터 연구소 </td><td> BK-2000 </td></tr><tr><td> L19 </td><td> Biotinylated Vicia 비오의 렉틴 </td><td> VVL </td><td> 벡터 연구소 </td><td> BK-2000 </td></tr><tr><td> L20 </td><td> Biotinylated Lycopersicon Esculentum (토마토) 렉틴 </td><td> LEL </td><td> 벡터 연구소 </td><td> BK-3000 </td></tr><tr><td> L21 </td><td> Biotinylated Ulex Europaeus Agglutinin 전 </td><td> UEA 전 </td><td> 벡터 연구소 </td><td> BK-1000 </td></tr><tr><td> L22 </td><td> Biotinylated Jacalin </td><td> JACALIN </td><td> 벡터 연구소 </td><td> BK-3000 </td></tr><tr><td> A1 </td><td> 염소 F (AB &#39;) 2 조각 반 인간 IgM, Fc5μ 항체 </td><td> IgM </td><td> 잭슨 Immuno 연구 </td><td> 109-006-129 </td></tr><tr><td> A2 </td><td> 돈키 F (AB &#39;) 2 Frag 안티 - 인간 IgG (H + L)tibody </td><td> AB1 </td><td> 잭슨 Immuno 연구 </td><td> 709-006-149 </td></tr><tr><td> A3 </td><td> 마우스 안티 인간 IgG F (AB &#39;) 2 단클론 항체 </td><td> AB3 </td><td> 잭슨 Immuno 연구 </td><td> 209-005-097 </td></tr><tr><td> A4 </td><td> 염소 안티 인간의 알파 2 macroglobulin polyclonal 항체 </td><td> A2M </td><td> GeneTex </td><td> GTX62924 </td></tr><tr><td> A5 </td><td> 토끼 반 인간 알파-1-antitrypsin polyclonal 항체 </td><td> A1AT </td><td> 리 Biosiences </td><td> CA1T-80A </td></tr><tr><td> A6 </td><td> 마우스 안티 인간 알파-1-antitrypsin 단클론 항체 </td><td> A1AT </td><td> 시그마 알드리치 </td><td> SAB4200198 </td></tr><tr><td> A7 </td><td> 토끼 반 인간 알파-1-antitrypsin polyclonal 항체 </td><td> 주문 </td><td> NeoMarkers </td><td> RB-367-A1 </td></tr><tr><td> A8 </td><td> 래빗 안티 인간알파-1-antichymotrypsin polyclonal 항체 </td><td> 주문 </td><td> 피셔 과학 </td><td> RB9213R7 </td></tr><tr><td> A9 </td><td> 마우스 안티 인간 트랜스페린 단클론 항체 </td><td> 트랜스페린 </td><td> GeneTex </td><td> GTX101035 </td></tr><tr><td> A10 </td><td> 토끼 반 인간 트랜스페린 polyclonal 항체 </td><td> 트랜스페린 </td><td> GeneTex </td><td> GTX77130 </td></tr><tr><td> A11 </td><td> 염소 안티 인간 apolipoprotein J polyclonal 항체 </td><td> ApoJ </td><td> Abcam </td><td> ab7610 </td></tr><tr><td> A12 </td><td> 마우스 안티 인간 GP73 단클론 항체 </td><td> GP73 </td><td> 애보트 </td><td> 14H4-23 </td></tr><tr><td> A13 </td><td> 마우스 안티 인간 GP73 단클론 항체 </td><td> GP73 </td><td> 산타 크루즈 생명 공학 INC </td><td> SC-101275 </td></tr><tr><td> A14 </td><td> 토끼 반 인간 알파-1 fetoprotein polyclonal 항체 </td><td> AFP </td><td> GenWay </td><td> GWB-41C966 </td></tr><tr><td> A15 </td><td> 마우스 안티 인간의 알파 페토 프로테인-1 단클론 항체 </td><td> AFP </td><td> 피츠제럴드 </td><td> 10-A05A </td></tr><tr><td> A16 </td><td> 마우스 안티 인간 hemopexin 단클론 항체 </td><td> Hemopexin </td><td> Assaypro </td><td> 60190-05011 </td></tr><tr><td> 대답 17 </td><td> 마우스 안티 인간 glypican-3 (1G12) 단클론 항체 </td><td> GPL3 </td><td> 산타 크루즈 바이오 </td><td> SC-65443 </td></tr><tr><td> 대답 18 </td><td> 마우스 안티 인간 Kininogen (LMW) 단클론 항체 </td><td> Kininogen </td><td> Assaypro </td><td> 20333-05011 </td></tr><tr><td> 대답 19 </td><td> 토끼 반 인간 MMP-21 단클론 항체 </td><td> MMP21 </td><td> Epitomic </td><td> 1955-1 </td></tr><tr><td> A20 </td><td> 마우스 안티 인간 CEACAM-1 단클론 antibodY </td><td> CEACAM </td><td> R &amp; D 시스템 </td><td> MAB1180 </td></tr><tr><td> 대답 21 </td><td> 쥐 안티 인간 DPPIV/CD26 단클론 항체 </td><td> DPPIV </td><td> R &amp; D 시스템 </td><td> MAB22441 </td></tr><tr><td> 대답 22 </td><td> 마우스 안티 인간 PIVKA II 단클론 항체 </td><td> PIVICA </td><td> 크리스탈 화학 </td><td> 8040 </td></tr><tr><td> 대답 23 </td><td> 마우스 안티 carcinoembryonic 항원 </td><td> CEA </td><td> 미국 생물학 </td><td> C1300 </td></tr><tr><td> 대답 24 </td><td> 마우스 방지 CA125 암 항원 </td><td> CA125 </td><td> 미국 생물학 </td><td> C0050-01D </td></tr><tr><td> 대답 25 </td><td> 마우스 방지 CA19-9 암 항원 </td><td> CA19-9 </td><td> 미국 생물학 </td><td> C0075-18 </td></tr><tr><td> 대답 26 </td><td> 마우스 방지 루이스 X 단클론 항체 </td><td> 루이스 X </td><td> Calbiochem </td><td> 434,631 </td></tr><tr><td> 바이오 </td><td> Biotinylated BSA (긍정적인 제어) </td><td> 바이오 </td><td> 집에서 만든 </td><td> N / A </td></tr></tbody></table> 이 프로토콜에서 사용 lectins 및 항체의 표 1.리스트. <table border="1"><tbody><tr><td> 시약 S / 장비의 명칭 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> 비 접촉 microarrayer </td><td> BioDot INC </td><td> sciFLEXARRAYER </td></tr><tr><td> 384 microplate </td><td> 어부 </td><td> 14-230-243 </td></tr><tr><td> FoodSaver </td><td> FoodSaver </td><td> V3835 </td></tr><tr><td> Ultrathin nitrocellulose coate microarray는 슬라이드 </td><td> Gentel </td><td> PATH </td></tr><tr><td> 슬라이드 Imprinter (옵션) </td><td> 젤 회사 </td><td> WSP60-1 </td></tr><tr><td> 흔드는 사람 </td><td> 어부 </td><td> 15-453-211 </td></tr><tr><td> 원심 분리기 </td><td> Eppendorf </td><td> 5804 000.013 </td></tr><tr><td> 이동식 랙과 슬라이드 씻는 세면대 / 슬라이드 염색법 요리 </td><td> 어부 </td><td> 08-812 </td></tr><tr><td> 슬라이드 배양 챔버 / 현미경 슬라이드 박스 </td><td> 어부 </td><td> 03-448-5 </td></tr><tr><td> Brij 35, 물 30 W / V % 용액 </td><td> Acros의 생명체 </td><td> AC32958-0025 </td></tr><tr><td> 십대 초반-20 </td><td> 어부 </td><td> P337-100 </td></tr><tr><td> 나트륨 Periodate (NaIO 4) </td><td> 시그마 </td><td> 311,448 </td></tr><tr><td> L-글루탐산 γ-히드라 지드 </td><td> 시그마 </td><td> G-7257 </td></tr><tr><td> 나트륨 아세테이트 무수 (CH 3 COONa) </td><td> 시그마 </td><td> S2889 </td></tr><tr><td> 소 혈청 알부민 (BSA) </td><td> Lampire 생물학 실험실 </td> &lt;TD&gt; 7,500,804 </td></tr><tr><td> 인산 버퍼 식염수 (PBS) (10X) </td><td> Denville 과학 </td><td> CP4390-48 </td></tr><tr><td> Dylight 549 복합 NeutrAvidin </td><td> 써모 </td><td> 22,837 </td></tr><tr><td> 테아제 억제제 칵테일 정제 </td><td> 로슈 </td><td> 4693159001 </td></tr><tr><td> ChromPure 인간 IgG, FC 조각 </td><td> 잭슨 Immunoresearch </td><td> 009-000-008 </td></tr><tr><td> ChromPure 인간 IgG, 전체 분자 </td><td> 잭슨 Immunoresearch </td><td> 009-000-003 </td></tr><tr><td> ChromPure 마우스 IgG, 전체 분자 </td><td> 잭슨 Immunoresearch </td><td> 015-000-003 </td></tr><tr><td> ChromPure 마우스 IgG, FC 조각 </td><td> 잭슨 Immunoresearch </td><td> 015-000-008 </td></tr><tr><td> ChromPure 토끼 IgG, 전체 분자 </td><td> 잭슨 Immunoresearch </td><td> 011-000-003 </td></tr><tr><td> ChromPure 돈키 IgG, 전체 분자 </td><td> 잭슨 Immunoresearch </td><td> 017-000-003 </td></tr><tr><td> Microarray 스캐너 </td><td> Tecan </td><td> LS는 리로디드 </td></tr></tbody></table> 이 프로토콜에 사용되는 장비와 시약의 표 2.리스트.

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.

Watch this Scientific Journal Video about Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples at JoVE.com

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

본 연구에서 우리는 특정 단백질의 글리코 실화 프로 파일링에서 사용할 수있는 렉틴 검출 방법과 멀티 플렉스 높은 처리량 항체 microarray를위한 향상된 프로토콜을 설명합니다. 이 프로토콜은 새로운 신뢰할 수있는 시약을 갖추고 있으며 훨씬 이전 절차에 비해 시간, 비용, 연구 장비 요구 사항을 줄여줍니다.

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 ...

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Biology

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