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

Dette arbejde blev støttet af Institut for hepatitis og Virus Research.

1. Udskrive en Antistof Microarray for assayet <ol><li> Fortyndes alle antistoffer til 0,5 mg / ml i phosphatbufret saltvand, pH 7,2 (PBS). </li><li> Portion 40 ul af hvert antistof i 384-brønds kilde plade. </li><li> Læg 384-brønds source plade på Scienion sciFLEXARRAYER microarrayer. </li><li> Læg 20 PATH microarray glider på microarrayer som mål. </li><li> Indstille microarrayer at udskrive 48 identiske subarrays, hvor 27-antistoffer og kontrol proteiner plettede in triplo i en 9x9 mønster (fig. 1E,...

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1. Target protein og capture Antibody Selection Forud for antistoffet mikroarray assayet nogle reagenser og materialer for at blive betragtet og fremstilles. At designe et antistof microarray for glycan profilering eller glycan biomarkør screening, et panel af antistoffer specifikke for glycoprotein ansøgere skal fastlægges i henhold til litteraturen eller fra tidligere resultater. Disse antistoffer var normalt købt fra forskellige leverandører, såsom R &amp; D Systems mv IgG foretrækkes...

<table border="1"><tbody><tr><td> ID </td><td> Navn på reagenset </td><td> Forkortelse </td><td> Firma </td><td> Katalog # </td></tr><tr><td> L1 </td><td> Biotinyleret Concanavalin A </td><td> ConA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L2 </td><td> Biotinyleret Sambucus nigra Lectin </td><td> SNA </td><td> Vector Laboratories </td><td> B-1305 </td></tr><tr><td> L3 </td><td> Biotinyleret Lens culinaris agglutinin </td><td> LCA </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L4 </td><td> Biotinyleret Ricinus communis agglutinin I </td><td> RCA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L5 </td><td> Biotinyleret Aleuria Aurantia Lectin </td><td> AAL </td><td> Vector Laboratories </td><td> B-1395 </td></tr><tr><td> L6 </td><td> Biotinyleret Erythrina Cristagalli Lectin </td><td> ECL </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L7 </td><td> Biotinyleret Griffonia (Bandeiraea) simplicifolia lectin II </td><td> GSL II </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L8 </td><td> Biotinyleret hvedekimagglutinin </td><td> WGA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L9 </td><td> Biotinyleret Phaseolus vulgaris Erythroagglutinin </td><td> PHA-E </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L10 </td><td> Biotinyleret Phaseolus vulgaris Leucoagglutinin </td><td> PHA-L </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L11 </td><td> Biotinyleret jordnøddeagglutinin </td><td> PNA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L12 </td><td> Biotinyleret Pisum sativum agglutinin </td><td> PSA </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L13 </td><td> Biotinyleret Dolichos Biflorus agglutinin </td><td> DBA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L14 </td><td> Biotinyleret Datura stramonium Lectin </td><td> DSL </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L15 </td><td> Biotinyleret Sophora Japonica agglutinin </td><td> SJA </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L16 </td><td> Biotinyleret Soybean agglutinin </td><td> SBA </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L17 </td><td> Biotinyleret Solanum tuberosum (Kartoffel) Lectin </td><td> STL </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L18 </td><td> Biotinyleret Griffonia (Bandeiraea) simplicifolia lectin I </td><td> GSL jeg </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L19 </td><td> Biotinyleret Vicia villosa Lectin </td><td> VVL </td><td> Vector Laboratories </td><td> BK-2000 </td></tr><tr><td> L20 </td><td> Biotinyleret Lycopersicon esculentum (Tomat) Lectin </td><td> LEL </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> L21 </td><td> Biotinyleret Ulex Europaeus agglutinin jeg </td><td> UEA jeg </td><td> Vector Laboratories </td><td> BK-1000 </td></tr><tr><td> L22 </td><td> Biotinyleret brødfrugt </td><td> Brødfrugt </td><td> Vector Laboratories </td><td> BK-3000 </td></tr><tr><td> A1 </td><td> Ged F (ab &#39;) 2 fragment-anti-humant IgM, Fc5μ antistof </td><td> IgM </td><td> Jackson Immuno Research </td><td> 109-006-129 </td></tr><tr><td> A2 </td><td> Æsel 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> Muse anti-human IgG F (ab &#39;) 2 monoklonalt antistof </td><td> ÆB3 </td><td> Jackson Immuno Research </td><td> 209-005-097 </td></tr><tr><td> A4 </td><td> Gede-anti-humant alpha 2 macroglobulin polyklonalt antistof </td><td> A2m </td><td> GeneTex </td><td> GTX62924 </td></tr><tr><td> A5 </td><td> Kanin-anti-humant alpha-1-antitrypsin-polyklonalt antistof </td><td> A1AT </td><td> Lee Biosiences </td><td> CA1T-80A </td></tr><tr><td> A6 </td><td> Muse anti-human alpha-1-antitrypsin-monoklonalt antistof </td><td> A1AT </td><td> Sigma Aldrich </td><td> SAB4200198 </td></tr><tr><td> A7 </td><td> Kanin-anti-humant alpha-1-antitrypsin-polyklonalt antistof </td><td> ACT </td><td> NeoMarkers </td><td> RB-367-A1 </td></tr><tr><td> A8 </td><td> Kanin-anti-humanalpha-1-antichymotrypsin polyklonalt antistof </td><td> ACT </td><td> Fisher Scientific </td><td> RB9213R7 </td></tr><tr><td> A9 </td><td> Muse anti-human transferrin monoklonalt antistof </td><td> Transferrin </td><td> GeneTex </td><td> GTX101035 </td></tr><tr><td> A10 </td><td> Kanin-anti-humant transferrin polyklonalt antistof </td><td> Transferrin </td><td> GeneTex </td><td> GTX77130 </td></tr><tr><td> A11 </td><td> Gede-anti-humant apolipoprotein J polyklonalt antistof </td><td> ApoJ </td><td> Abcam </td><td> ab7610 </td></tr><tr><td> A12 </td><td> Muse anti-human GP73 monoklonalt antistof </td><td> GP73 </td><td> Abbott </td><td> 14H4-23 </td></tr><tr><td> A13 </td><td> Muse anti-human GP73 monoklonalt antistof </td><td> GP73 </td><td> Santa Cruz Biotechnology INC </td><td> sc-101.275 </td></tr><tr><td> A14 </td><td> Kanin-anti-humant alfa-1 fetoprotein polyklonalt antistof </td><td> AFP </td><td> GenWay </td><td> GWB-41C966 </td></tr><tr><td> A15 </td><td> Muse anti-human alpha-1 føtoprotein monoklonalt antistof </td><td> AFP </td><td> Fitzgerald </td><td> 10-A05A </td></tr><tr><td> A16 </td><td> Muse anti-human hæmopexin monoklonalt antistof </td><td> Hæmopexin </td><td> Assaypro </td><td> 60190-05011 </td></tr><tr><td> A17 </td><td> Muse anti-human glypican-3 (1G12) monoklonalt antistof </td><td> GPL3 </td><td> Santa Cruz Bio </td><td> sc-65.443 </td></tr><tr><td> A18 </td><td> Muse anti-human kininogen (LMW) monoklonalt antistof </td><td> Kininogen </td><td> Assaypro </td><td> 20333-05011 </td></tr><tr><td> A19 </td><td> Kanin-anti-human MMP-21 monoklonalt antistof </td><td> MMP21 </td><td> Epitomic </td><td> 1955-1 </td></tr><tr><td> A20 </td><td> Muse anti-human CEACAM-1 monoklonalt antistofferney </td><td> CEACAM </td><td> R &amp; D Systems </td><td> MAB1180 </td></tr><tr><td> A21 </td><td> Rotte-anti-humant DPPIV/CD26 monoklonalt antistof </td><td> DPPIV </td><td> R &amp; D Systems </td><td> MAB22441 </td></tr><tr><td> A22 </td><td> Muse anti-human Pivka II monoklonalt antistof </td><td> PIVICA </td><td> Crystal kemi </td><td> 8040 </td></tr><tr><td> A23 </td><td> Muse anti-carcinoembryonisk antigen </td><td> CEA </td><td> USA biologiske </td><td> C1300 </td></tr><tr><td> A24 </td><td> Muse-anti-CA125 cancerantigen </td><td> CA125 </td><td> USA biologiske </td><td> C0050-01D </td></tr><tr><td> A25 </td><td> Muse-anti-CA19-9 cancerantigen </td><td> CA19-9 </td><td> USA biologiske </td><td> C0075-18 </td></tr><tr><td> A26 </td><td> Muse-anti-Lewis X-monoklonalt antistof </td><td> Lewis X </td><td> Calbiochem </td><td> 434631 </td></tr><tr><td> bio </td><td> Biotinyleret BSA (positiv kontrol) </td><td> Bio </td><td> Hjem-made </td><td> N / A </td></tr></tbody></table> Tabel 1. Liste over lectiner og antistoffer, der anvendes i denne protokol. <table border="1"><tbody><tr><td> Reagensets navn, s / udstyr </td><td> Firma </td><td> Katalog nummer </td></tr><tr><td> Ikke kontakt microarrayer </td><td> BioDot Inc </td><td> sciFLEXARRAYER </td></tr><tr><td> 384 mikrotiterplade </td><td> Fisher </td><td> 14-230-243 </td></tr><tr><td> FoodSaver </td><td> FoodSaver </td><td> V3835 </td></tr><tr><td> Ultratynde nitrocellulose coate microarray glider </td><td> Gentel </td><td> PATH </td></tr><tr><td> Slide Imprinter (valgfrit) </td><td> Gelen Company </td><td> WSP60-1 </td></tr><tr><td> Shaker </td><td> Fisher </td><td> 15-453-211 </td></tr><tr><td> Centrifuger </td><td> Eppendorf </td><td> 5804 000.013 </td></tr><tr><td> Slide håndvask / Slide Farvning Fad med aftagelig rack </td><td> Fisher </td><td> 08-812 </td></tr><tr><td> Objektglas rugekammeret / mikroskoppræparatglas box </td><td> Fisher </td><td> 03-448-5 </td></tr><tr><td> Brij 35, 30% w / v opløsning i vand </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 (NalO4) </td><td> Sigma </td><td> 311448 </td></tr><tr><td> L-glutaminsyre γ-hydrazid </td><td> Sigma </td><td> G-7257 </td></tr><tr><td> Natriumacetat Vandfri (CH3COONa) </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> Phosphatpuffersaltopløsning (PBS) (10X) </td><td> Denville Videnskabelige </td><td> CP4390-48 </td></tr><tr><td> Dylight 549 konjugeret NeutrAvidin </td><td> Thermo </td><td> 22837 </td></tr><tr><td> Proteaseinhibitor-cocktail 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, helt molekyle </td><td> Jackson Immunoresearch </td><td> 009-000-003 </td></tr><tr><td> ChromPure mus IgG, helt molekyle </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, helt molekyle </td><td> Jackson Immunoresearch </td><td> 011-000-003 </td></tr><tr><td> ChromPure Donkey IgG, helt molekyle </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> Tabel 2. Liste over udstyr og reagenser, der anvendes i denne protokol.

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 denne undersøgelse beskriver vi en forbedret protokol til et multiplekset high-throughput-antistof mikroarray med lectin påvisningsmetode, der kan anvendes i glycosylering profilering af specifikke proteiner. Denne protokol indeholder nye pålidelige reagenser og reducerer den tid, omkostninger og lab krav til udstyr i forhold til den tidligere procedure.

Kemisk blokeret antistof Microarray for Multiplexed Højkapacitetsforskning Profilering af specifikke protein Glycosylering i komplekse prøver

 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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