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

Este trabalho foi financiado pelo Instituto de Pesquisa de Vírus Hepatite e.

1. Imprimir um microarray de anticorpos para o Ensaio <ol><li> Diluir todos os anticorpos para 0,5 mg / ml em solução salina de tampão fosfato, pH 7,2 (PBS). </li><li> Alíquota 40 uL de cada anticorpo na placa de fonte 384 poços. </li><li> Coloque a placa de fonte de 384-bem para o microarrayer sciFLEXARRAYER Scienion. </li><li> Carregar 20 lâminas de microarray PATH para o microarrayer como alvo. </li><li> Defina o microarrayer para imprimir 48 subarrays idênticas, em que 27 anticorpos e proteínas de controle...

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1. Proteína alvo e seleção anticorpo de captura Antes do ensaio microarray anticorpo, alguns reagentes e materiais são necessários para ser considerado e preparado. Para conceber um microarray anticorpo para glicano perfil ou rastreio de glicano biomarcador, um painel de anticorpos específicos para os candidatos de glicoproteína deve ser determinada de acordo com a literatura ou a partir dos resultados anteriores. Esses anticorpos foram normalmente compradas de fornecedores diferentes, t...

<table border="1"><tbody><tr><td> ID </td><td> Nome do reagente </td><td> Abreviatura </td><td> Companhia </td><td> Catalogo </td></tr><tr><td> L1 </td><td> Concanavalina A biotinilado </td><td> ConA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L2 </td><td> Biotinilado Sambucus Nigra Lectina </td><td> SNA </td><td> Vector Laboratories </td><td> B-1305 </td></tr><tr><td> L3 </td><td> Biotinilado Lens culinaris aglutinina </td><td> LCA </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L4 </td><td> Biotinilado Ricinus communis aglutinina eu </td><td> RCA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L5 </td><td> Biotinilado Aleuria Lectina Aurantia </td><td> AAL </td><td> Vector Laboratories </td><td> B-1395 </td></tr><tr><td> L6 </td><td> Biotinilado Erythrina Lectina Cristagalli </td><td> ECL </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L7 </td><td> Biotinilado Griffonia (Bandeiraea) Lectina Simplicifolia II </td><td> GSL II </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L8 </td><td> Biotinilado Gérmen de Trigo aglutinina </td><td> WGA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L9 </td><td> Erythroagglutinin vulgaris biotinilado Phaseolus </td><td> PHA-E </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L10 </td><td> Leucoagglutinin vulgaris biotinilado Phaseolus </td><td> PHA-L </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L11 </td><td> Aglutinina de amendoim biotinilado </td><td> PNA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L12 </td><td> Biotinilado Pisum sativum aglutinina </td><td> PSA </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L13 </td><td> Biotinilado Dolichos biflorus aglutinina </td><td> DBA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L14 </td><td> Lectina Stramonium biotinilado Datura </td><td> DSL </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L15 </td><td> Aglutinina Sophora Japonica biotinilado </td><td> SJA </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L16 </td><td> Aglutinina de soja biotinilado </td><td> SBA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L17 </td><td> Biotinilado Solanum tuberosum (Batata) Lectina </td><td> STL </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L18 </td><td> Biotinilado Griffonia (Bandeiraea) Simplicifolia Lectina I </td><td> Eu GSL </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L19 </td><td> Biotinilado Vicia Lectina Villosa </td><td> VVL </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L20 </td><td> Biotinilado Lycopersicon esculentum (tomate) Lectina </td><td> LEL </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L21 </td><td> Ulex biotinilado Europaeus aglutinina eu </td><td> UEA I </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L22 </td><td> Jacalina biotinilado </td><td> Jacalina </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> A1 </td><td> F cabra (ab &#39;) 2 Fragmento IgM anti-humana, o anticorpo Fc5μ </td><td> IgM </td><td> Jackson Immuno Research </td><td> 109-006-129 </td></tr><tr><td> A2 </td><td> Burro F (ab &#39;) 2 Frag anti-IgG humana (H + L) umtibody </td><td> AB1 </td><td> Jackson Immuno Research </td><td> 709-006-149 </td></tr><tr><td> A3 </td><td> De ratinho anti-IgG humana F (ab &#39;) 2 de anticorpo monoclonal </td><td> AB3 </td><td> Jackson Immuno Research </td><td> 209-005-097 </td></tr><tr><td> A4 </td><td> Cabra anti-humano alfa 2 macroglobulina anticorpo policlonal </td><td> A2M </td><td> GeneTex </td><td> GTX62924 </td></tr><tr><td> A5 </td><td> De coelho anti-anticorpo humano alfa-1-antitripsina policlonal </td><td> A1AT </td><td> Lee Biosiences </td><td> CA1T-80A </td></tr><tr><td> A6 </td><td> De ratinho anti-anticorpo humano alfa-1-antitripsina monoclonal </td><td> A1AT </td><td> Sigma Aldrich </td><td> SAB4200198 </td></tr><tr><td> A7 </td><td> De coelho anti-anticorpo humano alfa-1-antitripsina policlonal </td><td> ACT </td><td> Neomarkers </td><td> RB-367-A1 </td></tr><tr><td> A8 </td><td> De coelho anti-humanoalfa-1-antiquimotripsina anticorpo policlonal </td><td> ACT </td><td> Fisher Scientific </td><td> RB9213R7 </td></tr><tr><td> A9 </td><td> Rato anti-humana de anticorpo monoclonal de transferrina </td><td> Transferrina </td><td> GeneTex </td><td> GTX101035 </td></tr><tr><td> A10 </td><td> De coelho anti-anticorpo humano policlonal de transferrina </td><td> Transferrina </td><td> GeneTex </td><td> GTX77130 </td></tr><tr><td> A11 </td><td> Cabra anti-humano apolipoproteína anticorpo policlonal J </td><td> ApoJ </td><td> Abcam </td><td> ab7610 </td></tr><tr><td> A12 </td><td> Rato anti-humana de GP73 anticorpo monoclonal </td><td> GP73 </td><td> Abbott </td><td> 14H4-23 </td></tr><tr><td> A13 </td><td> Rato anti-humana de GP73 anticorpo monoclonal </td><td> GP73 </td><td> Santa Cruz Biotechnology, Inc. </td><td> sc-101.275 </td></tr><tr><td> A14 </td><td> De coelho anti-humano de alfa-1 fetoprotein anticorpo policlonal </td><td> AFP </td><td> Genway </td><td> GWB-41C966 </td></tr><tr><td> A15 </td><td> De ratinho anti-anticorpo humano de alfa-1 fetoproteína monoclonal </td><td> AFP </td><td> Fitzgerald </td><td> 10-A05A </td></tr><tr><td> A16 </td><td> Rato anti-humana de anticorpo monoclonal hemopexina </td><td> Hemopexina </td><td> Assaypro </td><td> 60190-05011 </td></tr><tr><td> A17 </td><td> Rato anti-humano glypican-3 (1G12) anticorpo monoclonal </td><td> GPL3 </td><td> Santa Cruz Bio </td><td> sc-65.443 </td></tr><tr><td> A18 </td><td> Rato anti-humano Cininogênio anticorpo (LMW) monoclonal </td><td> Cininogênio </td><td> Assaypro </td><td> 20333-05011 </td></tr><tr><td> A19 </td><td> De coelho anti-anticorpo humano MMP-21 monoclonal </td><td> MMP21 </td><td> Epitomic </td><td> 1955-1 </td></tr><tr><td> A20 </td><td> Rato anti-humano CEACAM-1 anticorpos monoclonaisy </td><td> CEACAM </td><td> R &amp; D Systems </td><td> MAB1180 </td></tr><tr><td> A21 </td><td> Rat anti-humano DPPIV/CD26 anticorpo monoclonal </td><td> DPPIV </td><td> R &amp; D Systems </td><td> MAB22441 </td></tr><tr><td> A22 </td><td> Rato anti-humano PIVKA anticorpo monoclonal II </td><td> PIVICA </td><td> Cristal chem </td><td> 8040 </td></tr><tr><td> A23 </td><td> Antigénio de ratinho anti-carcinoembriónico </td><td> CEA </td><td> EUA biológica </td><td> C1300 </td></tr><tr><td> A24 </td><td> Antigen de ratinho anti-CA125 Câncer </td><td> CA125 </td><td> EUA biológica </td><td> C0050-01D </td></tr><tr><td> A25 </td><td> Antigénio de ratinho anti-CA19-9 Câncer </td><td> CA19-9 </td><td> EUA biológica </td><td> C0075-18 </td></tr><tr><td> A26 </td><td> De ratinho anti-Lewis anticorpo monoclonal x </td><td> Lewis X </td><td> Calbiochem </td><td> 434631 </td></tr><tr><td> bio </td><td> Biotinilado BSA (controle positivo) </td><td> Bio </td><td> Home-made </td><td> N / A </td></tr></tbody></table> Lista Tabela 1. De lectinas e anticorpos utilizados neste protocolo. <table border="1"><tbody><tr><td> Nome do reagente s / equipamentos </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Microarrayer sem contato </td><td> BioDot Inc </td><td> sciFLEXARRAYER </td></tr><tr><td> 384 microplacas </td><td> Pescador </td><td> 14-230-243 </td></tr><tr><td> FoodSaver </td><td> FoodSaver </td><td> V3835 </td></tr><tr><td> Nitrocelulose Ultrathin Coate microarray desliza </td><td> Gentel </td><td> PATH </td></tr><tr><td> Deslize Imprinter (opcional) </td><td> A Companhia Gel </td><td> WSP60-1 </td></tr><tr><td> Sacudidor </td><td> Pescador </td><td> 15-453-211 </td></tr><tr><td> Centrifugar </td><td> Eppendorf </td><td> 5804 000.013 </td></tr><tr><td> Deslize prato de coloração de lavagem bacia / Slide com rack removível </td><td> Pescador </td><td> 08-812 </td></tr><tr><td> Deslize incubação caixa de lâminas câmara / microscópio </td><td> Pescador </td><td> 03-448-5 </td></tr><tr><td> Brij 35, 30% de solução w / v em água </td><td> Acros Organics </td><td> AC32958-0025 </td></tr><tr><td> Tween-20 </td><td> Pescador </td><td> P337-100 </td></tr><tr><td> Periodato de sódio (NaIO4) </td><td> Sigma </td><td> 311448 </td></tr><tr><td> Ácido L-glutâmico γ-hidrazida </td><td> Sigma </td><td> G-7257 </td></tr><tr><td> De acetato de sódio anidro (CH 3 COONa) </td><td> Sigma </td><td> S2889 </td></tr><tr><td> Albumina de soro bovino (BSA) </td><td> Lampire Biológica Labs </td> &lt;td&gt; 7500804 </td></tr><tr><td> Tampão fosfato salino (PBS) (10X) </td><td> Denville Científico </td><td> CP4390-48 </td></tr><tr><td> Dylight NeutrAvidin 549 conjugado </td><td> Thermo </td><td> 22837 </td></tr><tr><td> Protease Comprimidos Coquetel Inibidor </td><td> Roche </td><td> 4693159001 </td></tr><tr><td> ChromPure IgG humana, fragmento Fc </td><td> Jackson Immunoresearch </td><td> 009-000-008 </td></tr><tr><td> ChromPure IgG humana, molécula inteira </td><td> Jackson Immunoresearch </td><td> 009-000-003 </td></tr><tr><td> ChromPure IgG de rato, molécula inteira </td><td> Jackson Immunoresearch </td><td> 015-000-003 </td></tr><tr><td> ChromPure IgG de rato, fragmento Fe </td><td> Jackson Immunoresearch </td><td> 015-000-008 </td></tr><tr><td> ChromPure IgG de coelho, molécula inteira </td><td> Jackson Immunoresearch </td><td> 011-000-003 </td></tr><tr><td> ChromPure Donkey IgG, molécula inteira </td><td> Jackson Immunoresearch </td><td> 017-000-003 </td></tr><tr><td> Microarray Scanner </td><td> Tecan </td><td> LS Reloaded </td></tr></tbody></table> Lista Tabela 2. De equipamentos e reagentes utilizados neste protocolo.

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

Neste estudo, nós descrevemos um protocolo melhorado para um microarray anticorpo multiplexado de alto rendimento com o método de detecção de lectina que pode ser usado em perfil de glicosilação de proteínas específicas. Este protocolo apresenta novos reagentes fiáveis ​​e reduz significativamente o tempo, custo e requisitos de equipamento de laboratório, em comparação com o procedimento anterior.

Microarray de anticorpos quimicamente bloqueada para Multiplexed perfil de alta capacidade de glicosilação proteína específica em amostras complexas

 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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