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

Questo lavoro è stato sostenuto dall&#39;Istituto per l&#39;epatite e la ricerca del virus.

1. Stampare un Microarray dell&#39;anticorpo per il test <ol><li> Diluire tutti gli anticorpi a 0,5 mg / ml in soluzione salina tampone fosfato, pH 7,2 (PBS). </li><li> Aliquotare 40 pl di ciascun anticorpo nella piastra a 384 pozzetti sorgente. </li><li> Caricare la piastra a 384 pozzetti sorgente sul micro-distributore sciFLEXARRAYER Scienion. </li><li> Caricare 20 vetrini microarray PATH sul micro-distributore come bersaglio. </li><li> Impostare la micro-distributore per stampare 48 sottoarray identici, in cui sono...

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1. Proteina bersaglio e selezione anticorpo di cattura Prima del test microarray anticorpi, alcuni reagenti e materiali sono necessari per essere considerato e preparati. Per progettare un microarray per l&#39;analisi degli anticorpi glicano o biomarker di screening glicano, un pannello di anticorpi specifici per i candidati della glicoproteina deve essere determinato secondo la letteratura o dai risultati precedenti. Questi anticorpi sono di solito acquistati da fornitori diversi, quali R &amp...

<table border="1"><tbody><tr><td> ID </td><td> Nome del reattivo </td><td> Abbreviazione </td><td> Azienda </td><td> Catalogo # </td></tr><tr><td> L1 </td><td> Biotinilato Concanavalina A </td><td> ConA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L2 </td><td> Biotinilato Sambucus Nigra lectina </td><td> SNA </td><td> Vector Laboratories </td><td> B-1305 </td></tr><tr><td> L3 </td><td> Biotinilato Lens Culinaris agglutinina </td><td> LCA </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L4 </td><td> Biotinilato Ricinus Communis agglutinina I </td><td> RCA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L5 </td><td> Biotinilato Aleuria aurantia lectina </td><td> AAL </td><td> Vector Laboratories </td><td> B-1395 </td></tr><tr><td> L6 </td><td> Biotinilato Erythrina cristagalli lectina </td><td> ECL </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L7 </td><td> Biotinilato Griffonia (Bandeiraea) Simplicifolia lectina II </td><td> GSL II </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L8 </td><td> Biotinilato di Germe di Grano agglutinina </td><td> WGA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L9 </td><td> Biotinilato Erythroagglutinin Phaseolus vulgaris </td><td> PHA-E </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L10 </td><td> Biotinilato Leucoagglutinin Phaseolus vulgaris </td><td> PHA-L </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L11 </td><td> Peanut agglutinina biotinilato </td><td> PNA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L12 </td><td> Biotinilato Pisum Sativum agglutinina </td><td> PSA </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L13 </td><td> Biotinilato Dolichos biflorus agglutinina </td><td> DBA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L14 </td><td> Biotinilato lectina Datura Stramonium </td><td> DSL </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L15 </td><td> Biotinilato agglutinina Sophora Japonica </td><td> SJA </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L16 </td><td> Soia agglutinina biotinilato </td><td> SBA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L17 </td><td> Biotinilato Solanum tuberosum (patata) lectina </td><td> STL </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L18 </td><td> Biotinilato Griffonia (Bandeiraea) Simplicifolia I lectina </td><td> GSL I </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L19 </td><td> Biotinilato Vicia Villosa lectina </td><td> VVL </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L20 </td><td> Biotinilato Lycopersicon esculentum (pomodoro) lectina </td><td> LEL </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L21 </td><td> Biotinilato Ulex Europaeus agglutinina I </td><td> UEA I </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L22 </td><td> Biotinilato Jacalin </td><td> JACALIN </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> A1 </td><td> Capra F (ab &#39;) 2 Frammento anti-IgM umane, anticorpi Fc5μ </td><td> IgM </td><td> Jackson Immuno Research </td><td> 109-006-129 </td></tr><tr><td> A2 </td><td> Donkey F (ab &#39;) 2 Frag anti-IgG umane (H + L) unotibody </td><td> AB1 </td><td> Jackson Immuno Research </td><td> 709-006-149 </td></tr><tr><td> A3 </td><td> Topo anti-IgG umane F (ab &#39;) 2 anticorpo monoclonale </td><td> AB3 </td><td> Jackson Immuno Research </td><td> 209-005-097 </td></tr><tr><td> A4 </td><td> Capra anti-umano alfa 2 macroglobulina anticorpo policlonale </td><td> A2M </td><td> GeneTex </td><td> GTX62924 </td></tr><tr><td> A5 </td><td> Coniglio anti-umano alfa-1-antitripsina anticorpo policlonale </td><td> A1AT </td><td> Lee Biosiences </td><td> CA1T-80A </td></tr><tr><td> A6 </td><td> Topo anti-umano alfa-1-antitripsina anticorpo monoclonale </td><td> A1AT </td><td> Sigma Aldrich </td><td> SAB4200198 </td></tr><tr><td> A7 </td><td> Coniglio anti-umano alfa-1-antitripsina anticorpo policlonale </td><td> ACT </td><td> NeoMarkers </td><td> RB-367-A1 </td></tr><tr><td> A8 </td><td> Coniglio anti-umanoalfa-1-antichimotripsina anticorpo policlonale </td><td> ACT </td><td> Fisher Scientific </td><td> RB9213R7 </td></tr><tr><td> A9 </td><td> Topo anti-transferrina umana anticorpo monoclonale </td><td> Transferrina </td><td> GeneTex </td><td> GTX101035 </td></tr><tr><td> A10 </td><td> Coniglio anti-transferrina umana anticorpo policlonale </td><td> Transferrina </td><td> GeneTex </td><td> GTX77130 </td></tr><tr><td> A11 </td><td> Capra anti-umano apolipoproteina J anticorpo policlonale </td><td> ApoJ </td><td> Abcam </td><td> ab7610 </td></tr><tr><td> A12 </td><td> Topo anti-umano GP73 anticorpo monoclonale </td><td> GP73 </td><td> Abbott </td><td> 14H4-23 </td></tr><tr><td> A13 </td><td> Topo anti-umano GP73 anticorpo monoclonale </td><td> GP73 </td><td> Santa Cruz Biotechnology INC </td><td> sc-101275 </td></tr><tr><td> A14 </td><td> Coniglio anti-umano alfa-1 fetoprotein anticorpo policlonale </td><td> AFP </td><td> Genway </td><td> GWB-41C966 </td></tr><tr><td> A15 </td><td> Murino anti-umano alfa-1 fetoproteina anticorpo monoclonale </td><td> AFP </td><td> Fitzgerald </td><td> 10-A05A </td></tr><tr><td> A16 </td><td> Topo anti-umano emopessina anticorpo monoclonale </td><td> Emopessina </td><td> Assaypro </td><td> 60.190-05.011 </td></tr><tr><td> A17 </td><td> Murino anti-umano glypican-3 (1g12) anticorpo monoclonale </td><td> GPL3 </td><td> Santa Cruz Bio </td><td> sc-65443 </td></tr><tr><td> A18 </td><td> Topo anti-umano chininogeno (LMW) anticorpo monoclonale </td><td> Chininogeno </td><td> Assaypro </td><td> 20.333-05.011 </td></tr><tr><td> A19 </td><td> Coniglio anti-umano MMP-21 anticorpo monoclonale </td><td> MMP21 </td><td> Epitomic </td><td> 1955-1 </td></tr><tr><td> A20 </td><td> Topo anti-umano CEACAM-1 anticorpi monoclonaliy </td><td> CEACAM </td><td> R &amp; D Systems </td><td> MAB1180 </td></tr><tr><td> A21 </td><td> Ratto anti-umano DPPIV/CD26 anticorpo monoclonale </td><td> DPPIV </td><td> R &amp; D Systems </td><td> MAB22441 </td></tr><tr><td> A22 </td><td> Topo anti-umano PIVKA II anticorpo monoclonale </td><td> PIVICA </td><td> Cristallo chem </td><td> 8040 </td></tr><tr><td> A23 </td><td> Topo anti-antigene carcinoembrionario </td><td> CEA </td><td> USA biologico </td><td> C1300 </td></tr><tr><td> A24 </td><td> Topo Antigen anti-cancro CA125 </td><td> CA125 </td><td> USA biologico </td><td> C0050-01D </td></tr><tr><td> A25 </td><td> Topo anti-CA19-9 Cancer antigene </td><td> CA19-9 </td><td> USA biologico </td><td> C0075-18 </td></tr><tr><td> A26 </td><td> Topo anti-Lewis x anticorpo monoclonale </td><td> Lewis x </td><td> Calbiochem </td><td> 434631 </td></tr><tr><td> bio </td><td> Biotinilato BSA (controllo positivo) </td><td> Bio </td><td> Fatto in casa </td><td> N / A </td></tr></tbody></table> Tabella 1. Elenco delle lectine e anticorpi utilizzati in questo protocollo. <table border="1"><tbody><tr><td> Nome del reattivo, s / attrezzature </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Contatto micro-distributore non </td><td> BioDot Inc </td><td> sciFLEXARRAYER </td></tr><tr><td> 384 micropiastra </td><td> Pescatore </td><td> 14-230-243 </td></tr><tr><td> Foodsaver </td><td> Foodsaver </td><td> V3835 </td></tr><tr><td> Ultrasottile nitrocellulosa Coate microarray scivola </td><td> Gentel </td><td> PATH </td></tr><tr><td> Presentazione Imprinter (opzionale) </td><td> Il Gel Società </td><td> WSP60-1 </td></tr><tr><td> Shaker </td><td> Pescatore </td><td> 15-453-211 </td></tr><tr><td> Centrifuga </td><td> Eppendorf </td><td> 5804 000.013 </td></tr><tr><td> Diapositiva lavandino / Slide Dish colorazione con Rack estraibile </td><td> Pescatore </td><td> 08-812 </td></tr><tr><td> Diapositiva camera di incubazione / vetrino da microscopio box </td><td> Pescatore </td><td> 03-448-5 </td></tr><tr><td> Brij 35, 30 w / v% di soluzione in acqua </td><td> Acros Organics </td><td> AC32958-0025 </td></tr><tr><td> Tween-20 </td><td> Pescatore </td><td> P337-100 </td></tr><tr><td> Periodato di sodio (NAIO 4) </td><td> Sigma </td><td> 311448 </td></tr><tr><td> L-glutammico γ-idrazide </td><td> Sigma </td><td> G-7257 </td></tr><tr><td> Acetato di sodio anidro (CH 3 COONa) </td><td> Sigma </td><td> S2889 </td></tr><tr><td> Albumina sierica bovina (BSA) </td><td> Lampire Biological Labs </td> &lt;td&gt; 7500804 </td></tr><tr><td> Tampone fosfato salino (PBS) (10X) </td><td> Denville scientifico </td><td> CP4390-48 </td></tr><tr><td> Dylight 549 neutravidina coniugato </td><td> Thermo </td><td> 22837 </td></tr><tr><td> Inibitori della proteasi Cocktail Tablets </td><td> Roche </td><td> 4693159001 </td></tr><tr><td> ChromPure IgG umane, frammento Fc </td><td> Jackson ImmunoResearch </td><td> 009-000-008 </td></tr><tr><td> ChromPure IgG umane, molecola intera </td><td> Jackson ImmunoResearch </td><td> 009-000-003 </td></tr><tr><td> ChromPure mouse IgG, molecola intera </td><td> Jackson ImmunoResearch </td><td> 015-000-003 </td></tr><tr><td> ChromPure mouse IgG, frammento Fc </td><td> Jackson ImmunoResearch </td><td> 015-000-008 </td></tr><tr><td> ChromPure IgG di coniglio, molecola intera </td><td> Jackson ImmunoResearch </td><td> 011-000-003 </td></tr><tr><td> ChromPure Donkey IgG, molecola intera </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> Tabella 2. Elenco di attrezzature e reagenti utilizzati in questo protocollo.

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

In questo studio, si descrive un protocollo migliorato per un multiplato ad alta resa microarray anticorpo con metodo di rilevazione lectina che può essere utilizzato in profili glicosilazione di proteine ​​specifiche. Questo protocollo offre nuovi reagenti affidabili e riduce sensibilmente i tempi, i costi e alle attrezzature di laboratorio rispetto alla procedura precedente.

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

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

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