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

Bu çalışma Hepatit ve Virüs Araştırmaları Enstitüsü tarafından desteklenmiştir.

1. Test için bir antikor Mikroarray yazdırmak <ol><li> 0.5 mg / fosfat tampon salin, ph 7.2 (PBS) içinde ml tüm antikorları ile seyreltilir. </li><li> 384 kuyucuklu kaynak plakasına alikotu her antikor 40 ul. </li><li> Scienion sciFLEXARRAYER microarrayer üzerine 384-iyi kaynak plaka yükleyin. </li><li> Hedef olarak microarrayer üzerine 20 PATH mikroarray slaytlar yükleyin. </li><li> 27 antikorları ve kontrol proteinler 9x9 modeli (Şekil 1E, 1F) olarak üç nüsha olarak tespit edildiği 4...

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Biol. 520, 39-58 (2009).</li><li>Yue, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Prevalence+and+Nature+of+Glycan+Alterations+on+Specific+Proteins+in+Pancreatic+Cancer+Patients+Revealed+Using+Antibody-Lectin+Sandwich+Arrays.">The Prevalence and Nature of Glycan Alterations on Specific Proteins in Pancreatic Cancer Patients Revealed Using Antibody-Lectin Sandwich Arrays.</a> Molecular &#38; Cellular Proteomics. 8, 1697-1707 (2009).</li><li>Wolf-Yadlin, A., Sevecka, M., MacBeath, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+protein+function+and+signaling+using+protein+microarrays.">Dissecting protein function and signaling using protein microarrays.</a> Current Opinion in Chemical Biology. 13, 398-405 (2009).</li><li>Richard, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+as+Applied+to+Inherited+Metabolic+Diseases.">Proteomics as Applied to Inherited Metabolic Diseases.</a> Current Proteomics. 6, 140-153 (2009).</li><li>Nolen, B., Winans, M., Marrangoni, A., Lokshin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+tumor-associated+antigen+autoantibody+profiles+in+healthy+controls+detected+by+multiplex+bead-based+immunoassay.">Aberrant tumor-associated antigen autoantibody profiles in healthy controls detected by multiplex bead-based immunoassay.</a> Journal of Immunological Methods. 344, 116-120 (2009).</li><li>Kuno, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focused+Differential+Glycan+Analysis+with+the+Platform+Antibody-assisted+Lectin+Profiling+for+Glycan-related+Biomarker+Verification.">Focused Differential Glycan Analysis with the Platform Antibody-assisted Lectin Profiling for Glycan-related Biomarker Verification.</a> Molecular &#38; Cellular Proteomics. 8, 99-108 (2009).</li><li>Hsu, K. -. 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1. Hedef protein ve yakalama antikor seçimi Antikor mikroarray assay önce belli bir reaktifler ve malzemeler sayılabilir için gerekli ve hazırlanır. Glukanıdır profilleme veya glukanıdır biyomarker tarama, glikoprotein adaylara spesifik antikorların bir panel için bir antikor mikroarray tasarlamak için literatüre veya önceki sonuçlarından göre tespit edilmelidir. Bu antikorlar, genellikle, R &amp; D Systems, vb IgGs gibi farklı satıcılardan satın alınmıştır önceki tes...

<table border="1"><tbody><tr><td> Kimlik </td><td> Reaktif Adı </td><td> Kısaltma </td><td> Şirket </td><td> # Kataloğa </td></tr><tr><td> L1 </td><td> Biyotinile Concanavalin A </td><td> ConA </td><td> Vektör Laboratuvarları </td><td> BK-1000 </td></tr><tr><td> L2 </td><td> Tinile Sambucus Nigra Lektin </td><td> SNA </td><td> Vektör Laboratuvarları </td><td> B-1305 </td></tr><tr><td> L3 </td><td> Tinile Lens Culinaris Aglütinin </td><td> LCA </td><td> Vektör Laboratuvarları </td><td> BK-2000 </td></tr><tr><td> L4 </td><td> Ricinus Communis Aglütinin Ben biyotinile </td><td> RCA </td><td> Vektör Laboratuvarları </td><td> BK-1000 </td></tr><tr><td> L5 </td><td> Tinile Aleuria Aurantia Lektin </td><td> AAL </td><td> Vektör Laboratuvarları </td><td> B-1395 </td></tr><tr><td> L6 </td><td> Tinile Erythrina Cristagalli Lektin </td><td> ECL </td><td> Vektör Laboratuvarları </td><td> BK-3000 </td></tr><tr><td> L7 </td><td> Tinile Griffonia (Bandeiraea) Simplicifolia lektin II </td><td> GSL II </td><td> Vektör Laboratuvarları </td><td> BK-3000 </td></tr><tr><td> L8 </td><td> Tinile buğday tohumu Aglütinin </td><td> WGA </td><td> Vektör Laboratuvarları </td><td> BK-1000 </td></tr><tr><td> L9 </td><td> Tinile Phaseolus vulgaris Erythroagglutinin </td><td> PHA-E </td><td> Vektör Laboratuvarları </td><td> BK-2000 </td></tr><tr><td> L10 </td><td> Tinile Phaseolus vulgaris Leucoagglutinin </td><td> PHA-L </td><td> Vektör Laboratuvarları </td><td> BK-2000 </td></tr><tr><td> L11 </td><td> Tinile Fıstık Aglütinin </td><td> PNA </td><td> Vektör Laboratuvarları </td><td> BK-1000 </td></tr><tr><td> L12 </td><td> Pisum Sativum Aglütinin biyotinile </td><td> PSA </td><td> Vektör Laboratuvarları </td><td> BK-2000 </td></tr><tr><td> L13 </td><td> Tinile Dolichos tohumları biflorus Aglütinin </td><td> DBA </td><td> Vektör Laboratuvarları </td><td> BK-1000 </td></tr><tr><td> L14 </td><td> Tinile Datura stramonium Lektin </td><td> DSL </td><td> Vektör Laboratuvarları </td><td> BK-3000 </td></tr><tr><td> L15 </td><td> Tinile Sophora Japonica Aglütinin </td><td> SJA </td><td> Vektör Laboratuvarları </td><td> BK-2000 </td></tr><tr><td> L16 </td><td> Tinile Soya Aglütinin </td><td> SBA </td><td> Vektör Laboratuvarları </td><td> BK-1000 </td></tr><tr><td> L17 </td><td> Tinile Solanum Tuberosum (Patates) Lektin </td><td> STL </td><td> Vektör Laboratuvarları </td><td> BK-3000 </td></tr><tr><td> L18 </td><td> Tinile Griffonia (Bandeiraea) Simplicifolia lektin I </td><td> GSL I </td><td> Vektör Laboratuvarları </td><td> BK-2000 </td></tr><tr><td> L19 </td><td> Tinile Vicia villosa Lektin </td><td> VVL </td><td> Vektör Laboratuvarları </td><td> BK-2000 </td></tr><tr><td> L20 </td><td> Tinile Lycopersicon esculentum (Domates) Lektin </td><td> LEL </td><td> Vektör Laboratuvarları </td><td> BK-3000 </td></tr><tr><td> L21 </td><td> Tinile Ulex europaeus Aglütinin I </td><td> UEA I </td><td> Vektör Laboratuvarları </td><td> BK-1000 </td></tr><tr><td> L22 </td><td> Biyotinile Jacalin </td><td> JACALIN </td><td> Vektör Laboratuvarları </td><td> BK-3000 </td></tr><tr><td> A1 </td><td> Keçi F (ab &#39;) 2 Fragment anti-insan IgG, Fc5μ antikoru </td><td> IgM </td><td> Jackson Immuno Araştırma </td><td> 109-006-129 </td></tr><tr><td> A2 </td><td> Eşek F (ab &#39;) 2 Frag anti-insan IgG (H + L) birtibody </td><td> AB1 </td><td> Jackson Immuno Araştırma </td><td> 709-006-149 </td></tr><tr><td> A3 </td><td> Fare anti-insan IgG F (ab &#39;) 2 monoklonal antikor </td><td> AB3 </td><td> Jackson Immuno Araştırma </td><td> 209-005-097 </td></tr><tr><td> A4 </td><td> Keçi anti-insan alfa 2 makroglobulin poliklonal antikor </td><td> A2M </td><td> GeneTex </td><td> GTX62924 </td></tr><tr><td> A5 </td><td> Tavşan anti-insan alfa-1-antitripsin poliklonal antikor </td><td> A1AT </td><td> Lee Biosiences </td><td> CA1T-80A </td></tr><tr><td> A6 </td><td> Fare anti-insan alfa-1-antitripsin monoklonal antikor </td><td> A1AT </td><td> Sigma Aldrich </td><td> SAB4200198 </td></tr><tr><td> A7 </td><td> Tavşan anti-insan alfa-1-antitripsin poliklonal antikor </td><td> ACT </td><td> Boyandı </td><td> RB-367-A1 </td></tr><tr><td> A8 </td><td> Tavşan anti-insanalfa-1-antichymotrypsin poliklonal antikor </td><td> ACT </td><td> Fisher Scientific </td><td> RB9213R7 </td></tr><tr><td> A9 </td><td> Fare anti-insan transferin monoklonal antikor </td><td> Transferrin </td><td> GeneTex </td><td> GTX101035 </td></tr><tr><td> A10 </td><td> Tavşan anti-insan transferin poliklonal antikor </td><td> Transferrin </td><td> GeneTex </td><td> GTX77130 </td></tr><tr><td> A11 </td><td> Keçi anti-insan apolipoprotein J poliklonal antikor </td><td> APOJ </td><td> Abcam </td><td> ab7610 </td></tr><tr><td> A12 </td><td> Fare anti-insan GP73 monoklonal antikor </td><td> GP73 </td><td> Abbott </td><td> 14H4-23 </td></tr><tr><td> A13 </td><td> Fare anti-insan GP73 monoklonal antikor </td><td> GP73 </td><td> SANTA CRUZ BİYOTEKNOLOJİ A.Ş. </td><td> sc-101.275 </td></tr><tr><td> A14 </td><td> Tavşan anti-insan alfa-1 fetoprotein poliklonal antikor </td><td> AFP </td><td> GenWay </td><td> GWB-41C966 </td></tr><tr><td> A15 </td><td> Fare anti-insan alfa-1 fetoprotein monoklonal antikor </td><td> AFP </td><td> Fitzgerald </td><td> 10-A05A </td></tr><tr><td> A16 </td><td> Fare anti-insan hemopexin monoklonal antikor </td><td> Hemopexin </td><td> Assaypro </td><td> 60190-05011 </td></tr><tr><td> A17 </td><td> Fare anti-insan glypican-3 (1G12) monoklonal antikor </td><td> GPL3 </td><td> Santa Cruz Bio </td><td> sc-65.443 </td></tr><tr><td> A18 </td><td> Fare anti-insan Kininogen (LMW) monoklonal antikor </td><td> Kininogen </td><td> Assaypro </td><td> 20333-05011 </td></tr><tr><td> A19 </td><td> Tavşan anti-insan MMP-21 monoklonal antikor </td><td> MMP21 </td><td> Epitomic </td><td> 1955-1 </td></tr><tr><td> A20 </td><td> Fare anti-insan CEACAM-1 monoklonal antikorlarınıny </td><td> CEACAM </td><td> R &amp; D Systems </td><td> MAB1180 </td></tr><tr><td> A21 </td><td> Sıçan anti-insan DPPIV/CD26 monoklonal antikor </td><td> DPPIV </td><td> R &amp; D Systems </td><td> MAB22441 </td></tr><tr><td> A22 </td><td> Fare anti-insan PIVKA II monoklonal antikor </td><td> PIVICA </td><td> Kristal chem </td><td> 8040 </td></tr><tr><td> A23 </td><td> Fare anti-karsinoembriyonik antijen </td><td> CEA </td><td> ABD biyolojik </td><td> C1300 </td></tr><tr><td> A24 </td><td> Fare anti-CA125 Kanser Antijen </td><td> CA125 </td><td> ABD biyolojik </td><td> C0050-01D </td></tr><tr><td> A25 </td><td> Fare anti-CA19-9 Cancer antijeni </td><td> CA19-9 </td><td> ABD biyolojik </td><td> C0075-18 </td></tr><tr><td> A26 </td><td> Fare anti-Lewis x monoklonal antikor </td><td> Lewis X </td><td> Calbiochem </td><td> 434631 </td></tr><tr><td> bio </td><td> Tinile BSA (pozitif kontrol) </td><td> Bio </td><td> Ev yapımı </td><td> N / A </td></tr></tbody></table> Bu protokolde kullanılan lektinler ve antikorların Tablo 1. Listesi. <table border="1"><tbody><tr><td> Reaktif s / ekipmanları Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> Temassız microarrayer </td><td> BioDot A.Ş. </td><td> sciFLEXARRAYER </td></tr><tr><td> 384 mikroplaka </td><td> Balıkçı </td><td> 14-230-243 </td></tr><tr><td> FoodSaver </td><td> FoodSaver </td><td> V3835 </td></tr><tr><td> Ultrathin nitroselüloz coate mikroarray slaytlar </td><td> Gentel </td><td> PATH </td></tr><tr><td> Slayt Damgalayıcı (isteğe bağlı) </td><td> Jel Şirket </td><td> WSP60-1 </td></tr><tr><td> Shaker </td><td> Balıkçı </td><td> 15-453-211 </td></tr><tr><td> Santrifüj </td><td> Eppendorf </td><td> 5804 000.013 </td></tr><tr><td> Çıkarılabilir Rack ile Slayt yıkama lavabo / Slayt Boyama Bulaşık </td><td> Balıkçı </td><td> 08-812 </td></tr><tr><td> Slayt inkübasyon odası / mikroskop lamı kutusu </td><td> Balıkçı </td><td> 03-448-5 </td></tr><tr><td> Brij 35, su içinde 30 w / v% çözelti </td><td> Acros Organics </td><td> AC32958-0025 </td></tr><tr><td> Tween-20 </td><td> Balıkçı </td><td> P337-100 </td></tr><tr><td> Sodyum periodat (NaIO 4) </td><td> Sigma </td><td> 311448 </td></tr><tr><td> L-glutamik asit γ-hidrazit </td><td> Sigma </td><td> G-7257 </td></tr><tr><td> Sodyum Asetat Susuz (CH 3 COONa) </td><td> Sigma </td><td> S2889 </td></tr><tr><td> Bovine Serum Albumin (BSA) </td><td> Lampire Biyolojik Labs </td> &lt;td&gt; 7500804 </td></tr><tr><td> Fosfat Tampon Salin (PBS) (10X) </td><td> Denville Bilimsel </td><td> CP4390-48 </td></tr><tr><td> Dylight 549 konjuge NeutrAvidin </td><td> Thermo </td><td> 22837 </td></tr><tr><td> Proteaz İnhibitör Kokteyl Tabletler </td><td> Roche </td><td> 4693159001 </td></tr><tr><td> ChromPure İnsan IgG, Fc parçası </td><td> Jackson Immunoresearch </td><td> 009-000-008 </td></tr><tr><td> ChromPure İnsan IgG, tüm molekül </td><td> Jackson Immunoresearch </td><td> 009-000-003 </td></tr><tr><td> ChromPure Fare IgG, tüm molekül </td><td> Jackson Immunoresearch </td><td> 015-000-003 </td></tr><tr><td> ChromPure Fare IgG, Fc parçası </td><td> Jackson Immunoresearch </td><td> 015-000-008 </td></tr><tr><td> Tavşan ChromPure IgG, tüm molekül </td><td> Jackson Immunoresearch </td><td> 011-000-003 </td></tr><tr><td> ChromPure Donkey IgG, tüm molekül </td><td> Jackson Immunoresearch </td><td> 017-000-003 </td></tr><tr><td> Mikroarray Tarayıcı </td><td> Tecan </td><td> LS Reloaded </td></tr></tbody></table> Bu protokolde kullanılan ekipman ve reaktiflerin Tablo 2. Listesi.

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

Bu çalışmada, özel proteinlerin glikozilasyon profil kullanılabilir lektin saptama yöntemi ile çoklanır yüksek verim antikoru mikroarray için geliştirilmiş bir protokol açıklanmaktadır. Bu protokol, yeni, güvenilir reaktif özellikleri ve önemli ölçüde daha önceki prosedüre göre zaman, maliyet ve laboratuar ekipmanları gereksinimlerini azaltır.

Kompleks Örnekleri spesifik protein Glikozilasyon arasında Multiplexed Yüksek verim Profilleme için Kimyasal-bloke Antikor Mikroarray

 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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