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

Detta arbete stöddes av Institutet för hepatit och Virus Research.

1. Skriv en antikropp Microarray för analysen <ol><li> Späd alla antikroppar till 0,5 mg / ml i fosfatbuffrad saltlösning, pH 7,2 (PBS). </li><li> Alikvot 40 | il av varje antikropp i den 384-brunnars platta källa. </li><li> Ladda 384-brunnars källa plattan på Scienion sciFLEXARRAYER microarrayer. </li><li> Ladda 20 bilder PATH microarray på microarrayer som mål. </li><li> Ställ microarrayer att skriva ut 48 identiska subsystem, där 27 antikroppar och proteiner kontroll är fläckig i tre exemplar i ett 9x9 ...

<ol>
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1. Målprotein och infångningsantikropp valet Före antikroppen microarray analysen, vissa reagenser och material måste beaktas och förberedas. Att designa en antikropp microarray för glykanen profilering eller glykan biomarkör screening, en panel av antikroppar som är specifika för glykoprotein kandidater bör fastställas enligt litteraturen eller från tidigare resultat. Dessa antikroppar var vanligen köptes från olika leverantörer som R &amp; D Systems etc. IgG är att föredra in...

<table border="1"><tbody><tr><td> ID </td><td> Namnet på reagens </td><td> Förkortning </td><td> Företaget </td><td> Katalog # </td></tr><tr><td> L1 </td><td> Biotinylerad Concanavalin A </td><td> ConA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L2 </td><td> Biotinylerad Sambucus Nigra Lektin </td><td> SNA </td><td> Vector Laboratories </td><td> B-1305 </td></tr><tr><td> L3 </td><td> Biotinylerad Lens culinaris agglutinin </td><td> LCA </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L4 </td><td> Biotinylerad Ricinus communis agglutinin I </td><td> RCA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L5 </td><td> Biotinylerad Aleuria Aurantia Lektin </td><td> AAL </td><td> Vector Laboratories </td><td> B-1395 </td></tr><tr><td> L6 </td><td> Biotinylerad Erythrina cristagalli Lektin </td><td> ECL- </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L7 </td><td> Biotinylerad Griffonia (Bandeiraea) simplicifolia-lektin II </td><td> GSL II </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L8 </td><td> Biotinylerad vetegroddsagglutinin </td><td> WGA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L9 </td><td> Biotinylerad Phaseolus vulgaris Erythroagglutinin </td><td> PHA-E </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L10 </td><td> Biotinylerad Phaseolus vulgaris Leucoagglutinin </td><td> PHA-L </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L11 </td><td> Biotinylerad jordnötsagglutinin </td><td> PNA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L12 </td><td> Biotinylerad Pisum sativum agglutinin </td><td> PSA </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L13 </td><td> Biotinylerad Dolichos biflorus agglutinin </td><td> DBA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L14 </td><td> Biotinylerad Datura stramonium Lektin </td><td> DSL </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L15 </td><td> Biotinylerad Sophora Japonica agglutinin </td><td> SJA </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L16 </td><td> Biotinylerad Soybean agglutinin </td><td> SBA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L17 </td><td> Biotinylerad Solanum tuberosum (potatis) Lektin </td><td> STL </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L18 </td><td> Biotinylerad Griffonia (Bandeiraea) simplicifolia-lektin I </td><td> GSL I </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L19 </td><td> Biotinylerad Vicia villosa Lektin </td><td> VVL </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L20 </td><td> Biotinylerad Lycopersicon esculentum (tomat) Lektin </td><td> LEL </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L21 </td><td> Biotinylerad Ulex Europaeus agglutinin I </td><td> UEA I </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L22 </td><td> Biotinylerad jakalin </td><td> Jacalin </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> A1 </td><td> Get F (ab &#39;) 2 fragment anti-humant IgM, Fc5μ antikropp </td><td> IgM </td><td> Jackson Immuno Research </td><td> 109-006-129 </td></tr><tr><td> A2 </td><td> Åsna F (ab &#39;) 2 Frag-anti-humant IgG (H + L) entibody </td><td> AB1 </td><td> Jackson Immuno Research </td><td> 709-006-149 </td></tr><tr><td> A3 </td><td> Mus-anti-human IgG F (ab &#39;) 2-monoklonal antikropp </td><td> AB3 </td><td> Jackson Immuno Research </td><td> 209-005-097 </td></tr><tr><td> A4 </td><td> Get-anti-human alfa 2-makroglobulin polyklonal antikropp </td><td> A2M </td><td> GeneTex </td><td> GTX62924 </td></tr><tr><td> A5 </td><td> Kanin-anti-human alfa-1-antitrypsin-polyklonal antikropp </td><td> A1AT </td><td> Lee Biosiences </td><td> CA1T-80A </td></tr><tr><td> A6 </td><td> Mus-anti-human alfa-1-antitrypsin-monoklonal antikropp </td><td> A1AT </td><td> Sigma Aldrich </td><td> SAB4200198 </td></tr><tr><td> A7 </td><td> Kanin-anti-human alfa-1-antitrypsin-polyklonal antikropp </td><td> AGERA </td><td> NeoMarkers </td><td> RB-367-A1 </td></tr><tr><td> A8 </td><td> Kanin-anti-humanalfa-1-antikymotrypsin polyklonal antikropp </td><td> AGERA </td><td> Fisher Scientific </td><td> RB9213R7 </td></tr><tr><td> A9 </td><td> Mus-anti-humant transferrin monoklonal antikropp </td><td> Transferrin </td><td> GeneTex </td><td> GTX101035 </td></tr><tr><td> A10 </td><td> Kanin-anti-humant transferrin polyklonal antikropp </td><td> Transferrin </td><td> GeneTex </td><td> GTX77130 </td></tr><tr><td> A11 </td><td> Get-anti-human apolipoprotein J polyklonal antikropp </td><td> ApoJ </td><td> Abcam </td><td> ab7610 </td></tr><tr><td> A12 </td><td> Mus-anti-human GP73 monoklonal antikropp </td><td> GP73 </td><td> Abbott </td><td> 14H4-23 </td></tr><tr><td> A13 </td><td> Mus-anti-human GP73 monoklonal antikropp </td><td> GP73 </td><td> Santa Cruz Biotechnology Inc </td><td> sc-101.275 </td></tr><tr><td> A14 </td><td> Kanin-anti-human alfa-1 fetoprotein polyklonal antikropp </td><td> AFP </td><td> Genway </td><td> GWB-41C966 </td></tr><tr><td> A15 </td><td> Mus-anti-human alfa-1 fetoprotein monoklonal antikropp </td><td> AFP </td><td> Fitzgerald </td><td> 10-A05A </td></tr><tr><td> A16 </td><td> Mus-anti-human hemopexin monoklonal antikropp </td><td> Hemopexin </td><td> Assaypro </td><td> 60.190-05.011 </td></tr><tr><td> A17 </td><td> Mus-anti-human glypican-3 (1G12) monoklonal antikropp </td><td> GPL3 </td><td> Santa Cruz Bio </td><td> sc-65.443 </td></tr><tr><td> A18 </td><td> Mus-anti-human kininogen (LMW) monoklonal antikropp </td><td> Kininogen </td><td> Assaypro </td><td> 20.333-05.011 </td></tr><tr><td> A19 </td><td> Kanin-anti-human MMP-21 monoklonal antikropp </td><td> MMP21 </td><td> Epitomic </td><td> 1955-1 </td></tr><tr><td> A20 </td><td> Mus-anti-human CEACAM-1 monoklonal antikroppar moty </td><td> CEACAM </td><td> R &amp; D Systems </td><td> MAB1180 </td></tr><tr><td> A21 </td><td> Råtta anti-human-DPPIV/CD26 monoklonal antikropp </td><td> DPPIV </td><td> R &amp; D Systems </td><td> MAB22441 </td></tr><tr><td> A22 </td><td> Mus-anti-human PIUKA II monoklonal antikropp </td><td> PIVICA </td><td> Kristall Chem </td><td> 8040 </td></tr><tr><td> A23 </td><td> Mus-anti-karcinoembryonalt antigen </td><td> CEA </td><td> US biologiska </td><td> C1300 </td></tr><tr><td> A24 </td><td> Mus-anti-CA125 cancerantigen </td><td> CA 125 </td><td> US biologiska </td><td> C0050-01D </td></tr><tr><td> A25 </td><td> Mus-anti-CA19-9 Cancer antigen </td><td> CA19-9 </td><td> US biologiska </td><td> C0075-18 </td></tr><tr><td> A26 </td><td> Mus-anti-Lewis x-monoklonal antikropp </td><td> Lewis X- </td><td> Calbiochem </td><td> 434.631 </td></tr><tr><td> bio </td><td> Biotinylerat BSA (positiv kontroll) </td><td> Bio </td><td> Home-made </td><td> N / A </td></tr></tbody></table> Tabell 1. Förteckning över lektiner och antikroppar som används i detta protokoll. <table border="1"><tbody><tr><td> Reagensets namn s / utrustning </td><td> Företaget </td><td> Katalognummer </td></tr><tr><td> Icke kontakt microarrayer </td><td> BioDot Inc </td><td> sciFLEXARRAYER </td></tr><tr><td> 384 mikroplatt </td><td> Fisher </td><td> 14-230-243 </td></tr><tr><td> FoodSaver </td><td> FoodSaver </td><td> V3835 </td></tr><tr><td> Ultratunna nitrocellulosa coate mikromatris glider </td><td> Gentel </td><td> VÄG </td></tr><tr><td> Skjut tryckverk (tillval) </td><td> Den Gel Företaget </td><td> WSP60-1 </td></tr><tr><td> Skakanordning </td><td> Fisher </td><td> 15-453-211 </td></tr><tr><td> Centrifugera </td><td> Eppendorf </td><td> 5804 000.013 </td></tr><tr><td> Slide tvätt bassäng / Slide Färgning skålen med Löstagbar Rack </td><td> Fisher </td><td> 08-812 </td></tr><tr><td> Slide inkubationskammaren / objektglas box </td><td> Fisher </td><td> 03-448-5 </td></tr><tr><td> Brij 35, 30 vikt / volym% lösning i vatten </td><td> Acros Organics </td><td> AC32958-0025 </td></tr><tr><td> Tween-20 </td><td> Fisher </td><td> P337-100 </td></tr><tr><td> Natriumperiodat (NalO 4) </td><td> Sigma </td><td> 311.448 </td></tr><tr><td> L-Glutaminsyra γ-hydrazid </td><td> Sigma </td><td> G-7257 </td></tr><tr><td> Natriumacetat Vattenfri (CH3 COONa) </td><td> Sigma </td><td> S2889 </td></tr><tr><td> Bovint serumalbumin (BSA) </td><td> Lampire Biologisk Labs </td> &lt;TD&gt; 7500804 </td></tr><tr><td> Fosfatbuffrad saltlösning (PBS) (10X) </td><td> Denville Scientific </td><td> CP4390-48 </td></tr><tr><td> Dylight 549 konjugerad neutravidin </td><td> Thermo </td><td> 22.837 </td></tr><tr><td> Proteasinhibitorcocktail Tabletter </td><td> Roche </td><td> 4693159001 </td></tr><tr><td> ChromPure Humant IgG, Fc-fragmentet </td><td> Jackson Immunoresearch </td><td> 009-000-008 </td></tr><tr><td> ChromPure Humant IgG, hel molekyl </td><td> Jackson Immunoresearch </td><td> 009-000-003 </td></tr><tr><td> ChromPure mus-IgG, hel molekyl </td><td> Jackson Immunoresearch </td><td> 015-000-003 </td></tr><tr><td> ChromPure mus-IgG, Fc-fragmentet </td><td> Jackson Immunoresearch </td><td> 015-000-008 </td></tr><tr><td> ChromPure Kanin-IgG, hel molekyl </td><td> Jackson Immunoresearch </td><td> 011-000-003 </td></tr><tr><td> ChromPure åsna-IgG, hel molekyl </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> Tabell 2. Förteckning över utrustning och reagenser som används i detta protokoll.

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

I denna studie beskriver vi ett förbättrat protokoll för en multiplexerad med hög genomströmning antikropp mikromatris med lektin detektionsmetod som kan användas i glykosylering profilering av specifika proteiner. Detta protokoll innehåller nya tillförlitliga reagenser och avsevärt minskar den tid, kostnad och lab krav på utrustning jämfört med föregående procedur.

Kemiskt blockerad antikropp Microarray för Multiplexade hög kapacitet profilering av specifik proteinglykosylering i komplexa prover

 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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