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

この作品は、肝炎やウイルス研究所によってサポートされていました。

1。アッセイ用の抗体マイクロアレイを印刷する<ol><li>リン酸緩衝生理食塩液、pH7.2（PBS）で0.5 mg / mlにすべての抗体を希釈する。 </li><li> 384ウェルソースプレートに各抗体のアリコートを40μl。 </li><li> Scienion sciFLEXARRAYERのマイクロアレイ上に384ウェルソースプレートをロードします。 </li><li>ターゲットとしてマイクロアレイ上20 PATHマイクロアレイスライドをロードします。 </li><li> 27抗体お?...

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1。標的タンパク質と捕捉抗体の選択抗体マイクロアレイアッセイに先立って、いくつかの試薬及び材料を考慮するために必要と用意されています。糖鎖プロファイリングや糖鎖バイオマーカーのスクリーニングのための抗体マイクロアレイを設計するには、糖タンパク質の候補に特異的な抗体のパネルは、文献に従って、または、以前の結果から応じて決定されるべきで?...

<table border="1"><tbody><tr><td> ID </td><td> 試薬の名前 </td><td> 略語 </td><td> 会社 </td><td> カタログ＃ </td></tr><tr><td> L1 </td><td>ビオチン化コンカナバリン</td><td> ConAを</td><td>ベクターラボラトリーズ</td><td> BK-1000 </td></tr><tr><td> L2 </td><td>ビオチン化したニワトコニグラレクチン</td><td> SNA </td><td>ベクターラボラトリーズ</td><td> B-1305 </td></tr><tr><td> L3 </td><td>ビオチン化したレンズマメレクチン</td><td> LCA </td><td>ベクターラボラトリーズ</td><td> BK-2000 </td></tr><tr><td> L4 </td><td>ビオチンヒマ凝集素I </td><td> RCA </td><td>ベクターラボラトリーズ</td><td> BK-1000 </td></tr><tr><td> L5 </td><td>ビオチン化したヒイロチャワンタケレクチン</td><td> AAL </td><td>ベクターラボラトリーズ</td><td> B-1395 </td></tr><tr><td> L6 </td><td>ビオチン化エリスリナ属Cristagalliレクチン</td><td> ECL </td><td>ベクターラボラトリーズ</td><td> BK-3000 </td></tr><tr><td> L7 </td><td>ビオチン化Griffonia（Bandeiraea）SimplicifoliaレクチンII </td><td> GSL II </td><td>ベクターラボラトリーズ</td><td> BK-3000 </td></tr><tr><td> L8 </td><td>ビオチン化した小麦胚芽凝集素</td><td> WGA </td><td>ベクターラボラトリーズ</td><td> BK-1000 </td></tr><tr><td> L9 </td><td>ビオチン化したインゲンマメのErythroagglutinin </td><td> PHA-E </td><td>ベクターラボラトリーズ</td><td> BK-2000 </td></tr><tr><td> L10 </td><td>ビオチン化したインゲンマメのLeucoagglutinin </td><td> PHA-L </td><td>ベクターラボラトリーズ</td><td> BK-2000 </td></tr><tr><td> L11 </td><td>ビオチン化ピーナッツ凝集素</td><td> PNA </td><td>ベクターラボラトリーズ</td><td> BK-1000 </td></tr><tr><td> L12 </td><td>ビオチン化エンドウレクチン</td><td> PSA </td><td>ベクターラボラトリーズ</td><td> BK-2000 </td></tr><tr><td> L13 </td><td>ビオチン化Dolichos Biflorus凝集</td><td> DBA </td><td>ベクターラボラトリーズ</td><td> BK-1000 </td></tr><tr><td> L14 </td><td>ビオチン化したチョウセン朝顔のレクチン</td><td> DSL </td><td>ベクターラボラトリーズ</td><td> BK-3000 </td></tr><tr><td> L15 </td><td>ビオチン化槐ジャポニカ凝集</td><td> SJA </td><td>ベクターラボラトリーズ</td><td> BK-2000 </td></tr><tr><td> L16 </td><td>ビオチン化ダイズ凝集素</td><td> SBA </td><td>ベクターラボラトリーズ</td><td> BK-1000 </td></tr><tr><td> L17 </td><td>ビオチン化したジャガイモ（ジャガイモ）レクチン</td><td> STL </td><td>ベクターラボラトリーズ</td><td> BK-3000 </td></tr><tr><td> L18 </td><td>ビオチン化Griffonia（バンドeiraea）SimplicifoliaレクチンI </td><td> GSL I </td><td>ベクターラボラトリーズ</td><td> BK-2000 </td></tr><tr><td> L19 </td><td>ビオチン化したソラマメVillosaレクチン</td><td> VVL </td><td>ベクターラボラトリーズ</td><td> BK-2000 </td></tr><tr><td> L20 </td><td>ビオチン標識トマトソバ（トマト）レクチン</td><td> LEL </td><td>ベクターラボラトリーズ</td><td> BK-3000 </td></tr><tr><td> L21 </td><td>ビオチン化Ulex Europaeus凝集素I </td><td> UEA I </td><td>ベクターラボラトリーズ</td><td> BK-1000 </td></tr><tr><td> L22 </td><td>ビオチン化ジャカリン</td><td>ジャカリン</td><td>ベクターラボラトリーズ</td><td> BK-3000 </td></tr><tr><td> A1 </td><td>ヤギF（ab &#39;）2フラグメント抗ヒトIgM、Fc5μ抗体</td><td> IgM抗体</td><td>ジャクソンイムノリサーチ</td><td> 109-006-129 </td></tr><tr><td> A2 </td><td>ロバのF（ab &#39;）2フラグメント抗ヒトIgG（H + L）tibody </td><td> AB1 </td><td>ジャクソンイムノリサーチ</td><td> 709-006-149 </td></tr><tr><td> A3 </td><td>マウス抗ヒトIgG F（ab &#39;）2のモノクローナル抗体</td><td> AB3 </td><td>ジャクソンイムノリサーチ</td><td> 209-005-097 </td></tr><tr><td> A4 </td><td>ヤギ抗ヒトα2マクログロブリンポリクローナル抗体</td><td> A2M </td><td> GeneTex </td><td> GTX62924 </td></tr><tr><td> A5 </td><td>ウサギ抗ヒトα-1アンチトリプシンポリクローナル抗体</td><td> A1AT </td><td>リーBiosiences </td><td> CA1T-80A </td></tr><tr><td> A6 </td><td>マウス抗ヒトα-1アンチトリプシンモノクローナル抗体</td><td> A1AT </td><td>シグマアルドリッチ</td><td> SAB4200198 </td></tr><tr><td> A7 </td><td>ウサギ抗ヒトα-1アンチトリプシンポリクローナル抗体</td><td> ACT </td><td> NeoMarkers </td><td> RB-367-A1 </td></tr><tr><td> A8 </td><td>ウサギ抗ヒトα-1-アンチキモトリプシンポリクローナル抗体</td><td> ACT </td><td>フィッシャー·サイエンティフィック</td><td> RB9213R7 </td></tr><tr><td> A9 </td><td>マウス抗ヒトトランスフェリンモノクローナル抗体</td><td>トランスフェリン</td><td> GeneTex </td><td> GTX101035 </td></tr><tr><td> A10 </td><td>ウサギ抗ヒトトランスフェリンポリクローナル抗体</td><td>トランスフェリン</td><td> GeneTex </td><td> GTX77130 </td></tr><tr><td> A11 </td><td>ヤギ抗ヒトアポリポタンパク質Jポリクローナル抗体</td><td> ApoJ </td><td>アブカム</td><td> ab7610 </td></tr><tr><td> A12 </td><td>マウス抗ヒトGP73モノクローナル抗体</td><td> GP73 </td><td>アボット</td><td> 14H4-23 </td></tr><tr><td> A13 </td><td>マウス抗ヒトGP73モノクローナル抗体</td><td> GP73 </td><td> SANTA CRUZ BIOTECHNOLOGY INC </td><td> SC-101275 </td></tr><tr><td> A14 </td><td>ウサギ抗ヒトα-1 fetoproteinポリクローナル抗体</td><td> AFP </td><td>建屋</td><td> GWB-41C966 </td></tr><tr><td> A15 </td><td>マウス抗ヒトα-1フェトプロテインモノクローナル抗体</td><td> AFP </td><td>フィッツジェラルド</td><td> 10-A05A </td></tr><tr><td> A16 </td><td>マウス抗ヒトヘモペキシンモノクローナル抗体</td><td>ヘモペキシン</td><td> Assaypro </td><td> 60190から05011 </td></tr><tr><td> A17 </td><td>マウス抗ヒトグリピカン3（1G12）モノクローナル抗体</td><td> GPL3 </td><td>サンタクルスバイオ</td><td> SC-65443 </td></tr><tr><td> A18 </td><td>マウス抗ヒトキニノゲン（LMW）モノクローナル抗体</td><td>キニノゲン</td><td> Assaypro </td><td> 20333から05011 </td></tr><tr><td> A19 </td><td>ウサギ抗ヒトMMP-21モノクローナル抗体</td><td> MMP21 </td><td> Epitomic </td><td> 1955から1 </td></tr><tr><td> A20 </td><td>マウス抗ヒトCEACAM-1モノクローナル不格好なやつY </td><td> CEACAM </td><td> R＆Dシステムズ</td><td> MAB1180 </td></tr><tr><td> A21 </td><td>ラット抗ヒトDPPIV/CD26モノクローナル抗体</td><td> DPPIV </td><td> R＆Dシステムズ</td><td> MAB22441 </td></tr><tr><td> A22 </td><td>マウス抗ヒトPIVKA IIモノクローナル抗体</td><td> PIVICA </td><td>結晶化学</td><td> 8040 </td></tr><tr><td> A23 </td><td>マウス抗癌胎児性抗原</td><td> CEA </td><td>米国の生物学</td><td> C1300 </td></tr><tr><td> A24 </td><td>マウス抗CA125癌抗原</td><td> CA125 </td><td>米国の生物学</td><td> C0050-01D </td></tr><tr><td> A25 </td><td>マウス抗CA19-9がん抗原</td><td> CA19-9 </td><td>米国の生物学</td><td> C0075-18 </td></tr><tr><td> A26 </td><td>マウス抗ルイスxモノクローナル抗体</td><td>ルイスX </td><td>カルビオケム</td><td> 434631 </td></tr><tr><td>バイオ</td><td>ビオチン化BSA（ポジティブコントロール） </td><td>バイオ</td><td>ホームメイド</td><td> N / A </td></tr></tbody></table>このプロトコルで使用されているレクチンと抗体の表1のリスト。 <table border="1"><tbody><tr><td> 試薬/ sの機器の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>非接触マイクロアレイ</td><td> BioDot株式会社</td><td> sciFLEXARRAYER </td></tr><tr><td> 384マイクロプレート</td><td>フィッシャー</td><td> 14-230-243 </td></tr><tr><td> FoodSaver </td><td> FoodSaver </td><td> V3835 </td></tr><tr><td>極薄のニトロセルロースcoateマイクロアレイスライド</td><td> Gentel </td><td>パス</td></tr><tr><td>スライドインプリンタ（オプション） </td><td>ゲルカンパニー</td><td> WSP60-1 </td></tr><tr><td>シェーカー</td><td>フィッシャー</td><td> 15-453-211 </td></tr><tr><td>遠心分離</td><td>エッペンドルフ</td><td> 5804 000.013 </td></tr><tr><td>リムーバブルラックスライド洗面/スライド染色皿</td><td>フィッシャー</td><td> 08から812 </td></tr><tr><td>スライドインキュベーションチャンバー/顕微鏡スライドボックス</td><td>フィッシャー</td><td> 03-448-5 </td></tr><tr><td> BRIJ 35、水に30 w / v％の溶液を</td><td>アクロスオーガニック</td><td> AC32958-0025 </td></tr><tr><td> Tween-20を</td><td>フィッシャー</td><td> P337-100 </td></tr><tr><td>過ヨウ素酸ナトリウム（過ヨウ素4） </td><td>シグマ</td><td> 311448 </td></tr><tr><td> L-グルタミン酸γ-ヒドラジド</td><td>シグマ</td><td> G-7257 </td></tr><tr><td>酢酸ナトリウム、無水（CH 3 COONa） </td><td>シグマ</td><td> S2889 </td></tr><tr><td>ウシ血清アルブミン（BSA） </td><td> Lampire生物学研究所</td> &lt;TD&gt; 7500804 </td></tr><tr><td>リン酸緩衝生理食塩水（PBS）（10X） </td><td>デンヴィル科学</td><td> CP4390-48 </td></tr><tr><td> Dylight 549コンジュゲートニュートラ</td><td>サーモ</td><td> 22837 </td></tr><tr><td>プロテアーゼインヒビターカクテル錠</td><td>ロシュ社</td><td> 4693159001 </td></tr><tr><td> ChromPureヒトIgG、Fcフラグメント</td><td>ジャクソンイムノ</td><td> 009-000-008 </td></tr><tr><td> ChromPureヒトIgG、分子全体</td><td>ジャクソンイムノ</td><td> 009-000-003 </td></tr><tr><td> ChromPureマウスIgG、分子全体</td><td>ジャクソンイムノ</td><td> 015-000-003 </td></tr><tr><td> ChromPureマウスIgG、Fcフラグメント</td><td>ジャクソンイムノ</td><td> 015-000-008 </td></tr><tr><td> ChromPureウサギIgG、分子全体</td><td>ジャクソンイムノ</td><td> 011-000-003 </td></tr><tr><td> ChromPureロバIgG抗体、分子全体</td><td>ジャクソンイムノ</td><td> 017-000-003 </td></tr><tr><td>マイクロアレイスキャナ</td><td>テカン</td><td> LSリローデッド</td></tr></tbody></table>このプロトコルで使用する器具や試薬の表2のリスト。

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

本研究では、特定のタンパク質のグリコシル化プロファイリングに使用することができるレクチン検出法による多重化、ハイスループット抗体マイクロアレイのための改良プロトコルを記述します。このプロトコルは、新しい信頼性の高い試薬を備えており、前の手順に比べて大幅に時間、コスト、およびラボ機器の要件を軽減します。

複雑なサンプル中の特定のタンパク質のグリコシル化の多重化ハイスループットプロファイリングのための化学的にブロックされた抗体マイクロアレイ

 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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