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

עבודה זו נתמכה על ידי המכון הפטיטיס ומחקר וירוס.

1. הדפס microarray נוגדן עבור assay <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לדלל את כל הנוגדנים ל 0.5 מ&quot;ג / מ&quot;ל ​​של תמיסת מלח פוספט חיץ, pH 7.2 (PBS). </li><li style=";text-align:right;direction:rtl"> Aliquot 40 μl של נוגדן זה לתוך צלחת 384, גם המקור. </li><li style=";text-align:right;direction:rtl"> טען את צלחת 384 גם מקור ?...

<ol>
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1. היעד חלבון נוגדן הבחירה ללכוד לפני assay microarray נוגדנים, חומרים כימיים וחומרים מסוימים יש צורך לקחת בחשבון ומוכן. לעצב microarray נוגדנים עבור פרופיל glycan או סמן ביולוגי glycan ההקרנה, פאנל של נוגדנים ספציפיים למועמדים גליקופרוטאין צריך להי...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> תעודת זהות </td><td> שם מגיב </td><td> קיצור </td><td> חברה </td><td> קטלוג # </td></tr><tr><td> L1 </td><td> Concanavalin Biotinylated </td><td> ConA </td><td> וקטור מעבדות </td><td> BK-1000 </td></tr><tr><td> L2 </td><td> Lectin Biotinylated Sambucus nigra </td><td> SNA </td><td> וקטור מעבדות </td><td> B-1305 </td></tr><tr><td> L3 </td><td> Biotinylated עדשה Culinaris Agglutinin </td><td> רע&quot;א </td><td> וקטור מעבדות </td><td> BK-2000 </td></tr><tr><td> L4 </td><td> Biotinylated Ricinus communis Agglutinin אני </td><td> RCA </td><td> וקטור מעבדות </td><td> BK-1000 </td></tr><tr><td> L5 </td><td> Lectin Biotinylated Aleuria Aurantia </td><td> אאל </td><td> וקטור מעבדות </td><td> B-1395 </td></tr><tr><td> L6 </td><td> Lectin Biotinylated Erythrina Cristagalli </td><td> ECL </td><td> וקטור מעבדות </td><td> BK-3000 </td></tr><tr><td> L7 </td><td> Biotinylated Griffonia (Bandeiraea) Simplicifolia Lectin II </td><td> GSL ב &#39; </td><td> וקטור מעבדות </td><td> BK-3000 </td></tr><tr><td> L8 </td><td> Biotinylated נבט חיטה Agglutinin </td><td> WGA </td><td> וקטור מעבדות </td><td> BK-1000 </td></tr><tr><td> L9 </td><td> Biotinylated vulgaris Phaseolus Erythroagglutinin </td><td> PHA-E </td><td> וקטור מעבדות </td><td> BK-2000 </td></tr><tr><td> L10 </td><td> Biotinylated vulgaris Phaseolus Leucoagglutinin </td><td> PHA-L </td><td> וקטור מעבדות </td><td> BK-2000 </td></tr><tr><td> L11 </td><td> בוטנים Biotinylated Agglutinin </td><td> הרשות הפלסטינית </td><td> וקטור מעבדות </td><td> BK-1000 </td></tr><tr><td> L12 </td><td> Biotinylated Pisum sativum Agglutinin </td><td> PSA </td><td> וקטור מעבדות </td><td> BK-2000 </td></tr><tr><td> L13 </td><td> Biotinylated לבלב Biflorus Agglutinin </td><td> DBA </td><td> וקטור מעבדות </td><td> BK-1000 </td></tr><tr><td> L14 </td><td> ב&quot;דאטורה Biotinylated וסטרמוניום Lectin </td><td> DSL </td><td> וקטור מעבדות </td><td> BK-3000 </td></tr><tr><td> L15 </td><td> Biotinylated Sophora Japonica Agglutinin </td><td> SJA </td><td> וקטור מעבדות </td><td> BK-2000 </td></tr><tr><td> L16 </td><td> סויה Biotinylated Agglutinin </td><td> SBA </td><td> וקטור מעבדות </td><td> BK-1000 </td></tr><tr><td> L17 </td><td> Biotinylated Solanum Tuberosum (תפוחי אדמה) Lectin </td><td> STL </td><td> וקטור מעבדות </td><td> BK-3000 </td></tr><tr><td> L18 </td><td> Biotinylated Griffonia (Bandeiraea) Simplicifolia Lectin אני </td><td> אני GSL </td><td> וקטור מעבדות </td><td> BK-2000 </td></tr><tr><td> L19 </td><td> Lectin Biotinylated בקיה Villosa </td><td> VVL </td><td> וקטור מעבדות </td><td> BK-2000 </td></tr><tr><td> L20 </td><td> Biotinylated Lycopersicon Esculentum (עגבניות) Lectin </td><td> לל </td><td> וקטור מעבדות </td><td> BK-3000 </td></tr><tr><td> L21 </td><td> Ulex Biotinylated Europaeus Agglutinin אני </td><td> אני UEA </td><td> וקטור מעבדות </td><td> BK-1000 </td></tr><tr><td> L22 </td><td> Biotinylated Jacalin </td><td> JACALIN </td><td> וקטור מעבדות </td><td> BK-3000 </td></tr><tr><td> A1 </td><td> F עיזים (&quot;א.ב.) 2 נגד אדם IgM שבר, נוגדנים Fc5μ </td><td> IgM </td><td> ג&#39;קסון חיסונית מחקר </td><td> 109-006-129 </td></tr><tr><td> A2 </td><td> החמור F (&#39;א.ב.) 2 Frag נגד אדם IgG (H + L)tibody </td><td> Ab1 </td><td> ג&#39;קסון חיסונית מחקר </td><td> 709-006-149 </td></tr><tr><td> A3 </td><td> עכבר נגד אדם IgG ו &#39;(&quot;א.ב.) 2 נוגדנים חד שבטיים </td><td> AB3 </td><td> ג&#39;קסון חיסונית מחקר </td><td> 209-005-097 </td></tr><tr><td> A4 </td><td> אנטי אנושי עז אלפא 2 macroglobulin polyclonal נוגדנים </td><td> A2M </td><td> GeneTex </td><td> GTX62924 </td></tr><tr><td> A5 </td><td> הארנב נגד אדם אלפא-1-antitrypsin polyclonal נוגדנים </td><td> A1AT </td><td> לי Biosiences </td><td> CA1T-80A </td></tr><tr><td> A6 </td><td> נגד אדם מאוס Alpha-1-antitrypsin נוגדנים חד שבטיים </td><td> A1AT </td><td> סיגמא אולדריץ&#39; </td><td> SAB4200198 </td></tr><tr><td> A7 </td><td> הארנב נגד אדם אלפא-1-antitrypsin polyclonal נוגדנים </td><td> מערכה </td><td> NeoMarkers </td><td> RB-367-A1 </td></tr><tr><td> A8 </td><td> הארנב נגד אדםAlpha-1-antichymotrypsin polyclonal נוגדנים </td><td> מערכה </td><td> פישר סיינטיפיק </td><td> RB9213R7 </td></tr><tr><td> A9 </td><td> נגד אדם מאוס transferrin חד שבטי נוגדן </td><td> Transferrin </td><td> GeneTex </td><td> GTX101035 </td></tr><tr><td> A10 </td><td> הארנב נגד אדם transferrin polyclonal נוגדנים </td><td> Transferrin </td><td> GeneTex </td><td> GTX77130 </td></tr><tr><td> A11 </td><td> אנטי אנושי עז אפוליפופרוטאין J polyclonal נוגדנים </td><td> ApoJ </td><td> Abcam </td><td> ab7610 </td></tr><tr><td> A12 </td><td> נגד אדם מאוס GP73 חד שבטי נוגדן </td><td> GP73 </td><td> אבוט </td><td> 14H4-23 </td></tr><tr><td> ת 13 </td><td> נגד אדם מאוס GP73 חד שבטי נוגדן </td><td> GP73 </td><td> סנטה קרוז ביוטכנולוגיה INC </td><td> SC-101275 </td></tr><tr><td> A14 </td><td> הארנב נגד אדם אלפא 1 fetoprotein polyclonal נוגדנים </td><td> AFP </td><td> GenWay </td><td> GWB-41C966 </td></tr><tr><td> ת 15 </td><td> נגד אדם מאוס Alpha-1 fetoprotein חד שבטי נוגדן </td><td> AFP </td><td> פיצג&#39;רלד </td><td> 10-A05A </td></tr><tr><td> A16 </td><td> נגד אדם מאוס hemopexin חד שבטי נוגדן </td><td> Hemopexin </td><td> Assaypro </td><td> 60190-05011 </td></tr><tr><td> A17 </td><td> עכבר נגד אדם glypican-3 (1G12) נוגדנים חד שבטיים </td><td> GPL3 </td><td> סנטה קרוז ביו </td><td> SC-65443 </td></tr><tr><td> A18 </td><td> נגד אדם מאוס Kininogen (LMW) נוגדנים חד שבטיים </td><td> Kininogen </td><td> Assaypro </td><td> 20333-05011 </td></tr><tr><td> A19 </td><td> הארנב נגד אדם MMP-21 חד שבטי נוגדן </td><td> MMP21 </td><td> Epitomic </td><td> 1955-1 </td></tr><tr><td> A20 </td><td> נגד אדם מאוס CEACAM-1 חד שבטי antibody </td><td> CEACAM </td><td> מחקר ופיתוח מערכות </td><td> MAB1180 </td></tr><tr><td> A21 </td><td> עכברוש נגד האדם נוגדנים חד שבטיים DPPIV/CD26 </td><td> DPPIV </td><td> מחקר ופיתוח מערכות </td><td> MAB22441 </td></tr><tr><td> A22 </td><td> נגד אדם מאוס PIVKA II נוגדנים חד שבטיים </td><td> PIVICA </td><td> קריסטל כימית </td><td> 8040 </td></tr><tr><td> A23 </td><td> נגד carcinoembryonic עכבר אנטיגן </td><td> CEA </td><td> ארה&quot;ב הביולוגי </td><td> C1300 </td></tr><tr><td> A24 </td><td> עכבר נגד CA125 סרטן Antigen </td><td> CA125 </td><td> ארה&quot;ב הביולוגי </td><td> C0050-01D </td></tr><tr><td> A25 </td><td> נגד CA19-9 סרטן עכבר אנטיגן </td><td> CA19-9 </td><td> ארה&quot;ב הביולוגי </td><td> C0075-18 </td></tr><tr><td> A26 </td><td> נגד לואיס עכבר x חד שבטי נוגדן </td><td> לואיס X </td><td> Calbiochem </td><td> 434631 </td></tr><tr><td> ביו </td><td> Biotinylated BSA (בקרה חיובית) </td><td> ביו </td><td> תוצרת בית </td><td> N / A </td></tr></tbody></table> 1. השולחן רשימת לקטינים ונוגדנים שימוש בפרוטוקול זה. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם / s מגיב ציוד </td><td> חברה </td><td> מספר קטלוגי </td></tr><tr><td> ללא מגע microarrayer </td><td> BioDot Inc </td><td> sciFLEXARRAYER </td></tr><tr><td> 384 microplate </td><td> דיג </td><td> 14-230-243 </td></tr><tr><td> FoodSaver </td><td> FoodSaver </td><td> V3835 </td></tr><tr><td> Ultrathin nitrocellulose coate microarray שקופיות </td><td> Gentel </td><td> PATH </td></tr><tr><td> Slide Imprinter (לא חובה) </td><td> החברה ג&#39;ל </td><td> WSP60-1 </td></tr><tr><td> מטרף </td><td> דיג </td><td> 15-453-211 </td></tr><tr><td> סרכזת </td><td> Eppendorf </td><td> 5804 000.013 </td></tr><tr><td> כביסה שקופיות אגן / שקופיות מכתים מהודרת עם מתלה נשלף </td><td> דיג </td><td> 08-812 </td></tr><tr><td> הדגירה שקופיות תא / מיקרוסקופ שקופיות תיבת </td><td> דיג </td><td> 03-448-5 </td></tr><tr><td> Brij 35, 30 W / V הפתרון% במים </td><td> Acros Organics </td><td> AC32958-0025 </td></tr><tr><td> Tween-20 </td><td> דיג </td><td> P337-100 </td></tr><tr><td> נתרן Periodate (NaIO 4) </td><td> סיגמא </td><td> 311448 </td></tr><tr><td> L-גלוטמית חומצה γ-hydrazide </td><td> סיגמא </td><td> G-7257 </td></tr><tr><td> אצטט וסודיום (CH 3 COONa) </td><td> סיגמא </td><td> S2889 </td></tr><tr><td> שור סרום אלבומין (BSA) </td><td> Lampire ביולוגית Labs </td> &lt;td&gt; 7500804 </td></tr><tr><td> מאגר מלוחים פוספט (PBS) (10X) </td><td> Denville מדעי </td><td> CP4390-48 </td></tr><tr><td> Dylight 549 מצומדות NeutrAvidin </td><td> Thermo </td><td> 22837 </td></tr><tr><td> מעכבי פרוטאז קוקטייל טבליות </td><td> רוש </td><td> 4693159001 </td></tr><tr><td> ChromPure האדם IgG, קטע Fc </td><td> ג&#39;קסון Immunoresearch </td><td> 009-000-008 </td></tr><tr><td> ChromPure לנוגדנים אנושיים, כל מולקולה </td><td> ג&#39;קסון Immunoresearch </td><td> 009-000-003 </td></tr><tr><td> ChromPure עכבר IgG, כל מולקולה </td><td> ג&#39;קסון Immunoresearch </td><td> 015-000-003 </td></tr><tr><td> ChromPure עכבר IgG, קטע Fc </td><td> ג&#39;קסון Immunoresearch </td><td> 015-000-008 </td></tr><tr><td> ChromPure הארנב IgG, כל מולקולה </td><td> ג&#39;קסון Immunoresearch </td><td> 011-000-003 </td></tr><tr><td> ChromPure חמור IgG, כל מולקולה </td><td> ג&#39;קסון Immunoresearch </td><td> 017-000-003 </td></tr><tr><td> Microarray סורק </td><td> טקאן </td><td> LS מחדש </td></tr></tbody></table> 2. לוח רשימת ציוד ריאגנטים שימוש בפרוטוקול זה.

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

במחקר זה, אנו מתארים פרוטוקול משופר עבור microarray תפוקה גבוהה מרובב נוגדנים עם שיטת הזיהוי lectin כי ניתן להשתמש פרופיל glycosylation של חלבונים מסוימים. פרוטוקול זה כולל ריאגנטים אמינים חדשים מפחית באופן משמעותי את הזמן, עלויות, ציוד מעבדה דרישות לעומת בהליך הקודם.

נוגדן microarray כימי חסומים פרופיל תפוקה גבוהה מרובב של Glycosylation חלבון מסוים בדגימות מורכבות

 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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