This work was supported by a grant from the NIH. The assays shown in Figure 1 was performed by Dr. Joseph Fasolo. We thank Dr. Rui Chen for helpful comments.

1. בדיקה הכנה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תרבות ולטהר את בדיקות קינאז V5-היתוך משמשות לבחינת אינטראקציות עם חלבונים אחרים כדלקמן: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> השתמש Y258 זן שמרים טריים מפוספס (מאטה pep4-3, his4-580, ura3-53, leu2-3,112) המכיל חלבון V5-היתוך (הביע מפאי-DEST52 GATEWAY הווקטור). השתמש בחלבון המבודד כבדיקה על microarrays. צלחת השמרים על שלם-אורציל הסינתטי (SC-Ura) / 2% דקסטרוז / אגר ולגדול על 30 מעלות צלזיוס במשך 3 ימים מתרבות קפוא (° גליצרול מניית -80 מעלות). </li><li style=";text-align:right;direction:rtl"> לחסן תרבויות המתנע (5-20 מיליליטר) ממושבה אחת ולגדול בין לילה בSC-Ura / 2% דקסטרוז על רועד פלטפורמה (220-250 סל&quot;ד) או גלגל על ​​30 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> למחרת בבוקר, לחסן 400 מיליליטר של SC-Ura 2% raffinose התרבות / עם תרבות המתנע מספיק כדי OD סופי 600 של 0.1. </li><li style=";text-align:right;direction:rtl"> לגדול inoculums לOD 600 של 0.6 ואחריו האינדוקציה גלקטוז של פו V5-קינאזשיאון לבנות ביטוי על ידי הוספת פתרון של תמצית שמרי 3x / Peptone (כן) בתוספת 6% גלקטוז, על ידי הוספה מספיק כדי לדלל את תקשורת האינדוקציה בפקטור של 3, כך שהריכוז הסופי של גלקטוז הוא 2%. </li><li style=";text-align:right;direction:rtl"> לגרום לתאים על 30 מעלות צלזיוס במשך 6 שעות על פלטפורמה רועדת. השתמש בבקבוק ליטר Erlenmeyer 2 כדי להבטיח אוורור מתאים. </li><li style=";text-align:right;direction:rtl"> קציר תאים באמצעות JA-10 (או דומה) הרוטור על ידי ספינינג 400 מיליליטר של השעיה תא XG ב 1000 במשך 5 דקות על 4 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> לשטוף את התאים פעם אחת עם 50 מיליליטר של קרח הקר PBS חיץ ולהעביר צינור חרוטי 50 מיליליטר. שטוף את הכדור שוב במאגר PBS קר כקרח (ללא חומרי ניקוי או תוספים אחרים) המשמשים לתמוגה והעברת 2 מיליליטר snap-כובע צינורות לתמוגה. </li><li style=";text-align:right;direction:rtl"> ספין התאים ב 20,000 XG דקות 1 ב 4 ° C לגלולה ופיפטה משם למאגר. </li><li style=";text-align:right;direction:rtl"> מניחים צינורות על קרח ולהמשיך בשלב תמוגה. </li><li style=";text-align:right;direction:rtl"> Lyse התאים (גלולה 250-350 μl) עם 0.5 מ&quot;מ חרוזים zirconia ב1: 1: 1 נפח תא גלולה, חרוזים, ונאגר מלוח פוספט (PBS) תמוגה חיץ ומערבולת התערובת באמצעות פלטפורמת תסיסה 3 פעמים ב 2 מרווחי דקות ב 4 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה lysate ב -20,000 XG בmicrofuge שולחן במשך 10 דקות ב 4 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> supernatant פיפטה לתוך צינור צנטריפוגות במהירות גבוהה פוליקרבונט ולהבהיר ידי ulracentrufugation למשך 30 דקות ב 150,000 XG ב 4 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> העבר את lysate הבהיר לצינור המכיל שרף prewashed Ni 2 + זיקה (~ 100 μl) דגירה על nutator לשעה 2 ב 4 ° C כדי ללכוד היסטידין 6X (שלו) מתויג חלבון V5-היתוך. </li><li style=";text-align:right;direction:rtl"> לשטוף את השרף שלוש פעמים במשך 10 דקות ב 4 מעלות צלזיוס עם הצפת לשטוף. גלולה השרף באמצעות צנטריפוגות בראש טבלה במשך 5 דקות XG ב 1000 ב 4 מעלות צלזיוס ולשאוב supernatant. הוסף חוצץ לשטוף טרי לכל אחד לשטוף ולהחזיר את הצינור לnutator תסיסה במהלך השטיפה. </li><li style=";text-align:right;direction:rtl"> לאחר STE לשטוףנ.ב. שלמים, להחיל את השרף שטף לעמודת G-25 טרי לelution. </li><li style=";text-align:right;direction:rtl"> החל 25 μl של חיץ elution באופן צעד חכם מתחיל עם 100 מ&quot;מ Imidazole 500 מ&quot;מ imidazole במרווחים של 50 מ&quot;מ עבור הסכום כולל של 9 שברים. </li><li style=";text-align:right;direction:rtl"> Assay השברים שנאספו באמצעות ג&#39;ל polyacrylamide סולפט dodecyl נתרן (SDS-עמוד) וצביעת coomassie לזהות את השבר (ים) המועשר הגבוהה ביותר לשימוש בassay ולהוסיף גליצרול 30% ולאחסן ב -80 ° C עד שימוש ב assay. </li></ol></li></ol> 2. גשוש מערכים הערה: אנא ראה שלב 2.6 סעיף זה כדי להכין את פתרון הנוגדנים לפני תחילת assay. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי לזהות אינטראקציות, לדלל את הנוגדן מצומדות V5-fluorophore (כלומר AlexaFluor 647) 260 / מיליליטר ng במאגר בדיקה ומערבבים היטב על ידי רועד. הערה: הכן פתרון 30 דקות לפני שימוש ומניח את הצינור על nutat זה נוגדןאו או גלגל כדי להבטיח ערבוב מלא והשעיה הומוגנית של נוגדן. </li><li style=";text-align:right;direction:rtl"> לדלל את הבדיקה חלבון V5-היתוך על פני טווח ריכוז של 5-500 מיקרוגרם / מיליליטר. הערה: אופטימלי עבור כל assay אינטראקציה בין חלבונים, באמצעות חיץ בדיקה. אופטימיזציה כוללת הוספה עוד בדיקות כדי לבחון חיץ. בדרך כלל 10 מיקרוגרם / מיליליטר משמש כנקודת התחלה ומותאמת בהתאם מבוסס על עוצמת האות. </li><li style=";text-align:right;direction:rtl"> הסר את microarrays החלבון מהמקפיא (-20 ° C) ומביא ל4 מעלות צלזיוס במקרר רק לפני השימוש. </li><li style=";text-align:right;direction:rtl"> להוסיף חיץ חסימה ישירות לבעל השקופית המכיל את חלבון micorarrays ולכסות את החלק העליון עם parafilm כדי למנוע דליפה. לחסום את מערכי חסימת חיץ במשך שעה 1 על ידי רועד ב 50 סל&quot;ד בשלב ב 4 ° C. </li><li style=";text-align:right;direction:rtl"> לאחר החסימה, להעביר את המערכים לתא humidified המקורר עד 4 ° C, ולהוסיף 90 μl של בדיקה דילול ישירות על פני המערך. כיסוי ARRays עם תלוש מרים גדל ודגירת סטטי (לא רועד) בתא humidified ב 4 מעלות צלזיוס במשך 1.5 שעות. </li><li style=";text-align:right;direction:rtl"> שטוף את המערכים 3 פעמים 1 דקות כל אחד בחיץ בדיקה בשלושה צינורות 50 מיליליטר חרוטי. הוסף את השקופיות לצינורות חרוטי המכילים מאגר בדיקה טרום צונן מספיק כדי המעטפה השקופית לחלוטין. לאפשר להחליק המרים להחליק בעדינות את של microarray החלבון (לא מכריח אותו כמו זה יכול לגרום נזק למשטח המערך). </li><li style=";text-align:right;direction:rtl"> החל פתרון הנוגדן ישירות למערך מייד לאחר שסיים את השטיפה (שלב 2.5) והשכבה עם תלוש מרים גדל כמו קודם. דגירה המערכים למשך 30 דקות ב 4 מעלות צלזיוס בתא humidified. </li><li style=";text-align:right;direction:rtl"> לבצע את אותה שלב לשטוף כמו קודם (דקות 3 פעמים 1 בחיץ בדיקה), וספין בצינור חרוטי 50 מיליליטר ב 800 XG בצנטריפוגה שולחן במשך 5 דקות בטמפרטורת חדר. אוויר יבש המערכים בהחזיק שקופיות בחושך במשך 30 דקות לפני סריקת המערך ב647 ננומטר. </li></ol>

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The authors declare that they have no competing financial interests.

הפרוטוקול שהוצג במקור מבוצע באמצעות 85 חלבוני היתוך קינאז-V5 שמרי חלבון ייחודיים כדי להשוות פעילות מחייבת בין משפחות שונות וקשורות של קינאזות שמרי חלבון וכתוצאה מכך ההזדהות של רשתות האינטראקציה קינאז החדשות in vivo 1. כטכנולוגית פרופיל proteomic מתעוררים, הפיתוח ש...

הצורך לבצע ניתוחים הגלובליים של ביוכימיה של חלבונים ומחייבים פעילות בvivo הביא לפיתוח שיטות החדשות לאפיון אינטראקציות בין חלבונים (PPIs) ולאחר translational השינויים של proteomes כל 1,3-8. microarrays חלבון מיוצרים כmicroarrays פונקציונלי חלבון באמצעות חלבונים פונקציונליים באורך מלא 4-6,8,9, או microarrays חלבון אנליטיים המכיל נוגדני 10,11. הם מתוכננים להכיל בצפיפות גבוהה של חלבונים ערוכים על גבי שקופיות מיקרוסקופ עם מגוון רחב של chemistries משטח כדי להקל על מגוון של תנאי ניסוי הנדרשים לביצוע ביוכימיים רחבים מנתחת 12. chemistries Nitrocellulose ומשטח אלדהיד לקובץ מצורף כימי באמצעות שיטות ליזין או קובץ מצורף זיקה כגון מגלשות chelated ניקל לחיבור חלבונים וגלוטתיון מתויג לקובץ מצורף זיקה בין יתר 13. </p&gt; השימוש בmicroarrays חלבון הפונקציונלי כדי לזהות אינטראקציות חלבון-חלבון דורש גישה לספריית חלבון פונקציונלי באיכות גבוהה 14. ס cerevisiae ניתנים להפקת ספרייה כגון דרך הזיווג של גבוה עותק זיקה מתויג מבני חלבון עם טכניקות טיהור chromatographic תפוקה גבוהה. הרוב המכריע של שמרי הגנום כבר רצף וכמעט כל proteome יכולה לבוא לידי ביטוי מגבוה עותק פלסמיד לטיהור וביוכימיים מנתחים 12. ברגע שהחלבונים מתקבלים וערוכים בפורמט 384 גם, הם מודפסים על גבי שקופית מיקרוסקופ המאפשרת ניתוח מהיר מקביל רב פרמטרית ביוכימיים וחקירה bioinformatic 8,14-16. microarrays החלבון שימש עבור מבחני האנזימטית ואינטראקציות עם חלבונים, שומנים, מולקולות קטנות, וחומצות גרעין בין יישומים רבים אחרים. הנגישות של חלבונים על פני השטח של proteomeמערכים להפוך אותם נוחים לסוגים שונים של זיהוי אנליטיים כוללים, חיסון-זיקה, משטח Plasmon תהודה, הקרינה ורבים טכניקות אחרות. יתר על כן, היא מאפשרת לבקרה טובה של תנאי הניסוי שבו זה יכול להיות קשה לעשות in vivo. המטרה של פרוטוקול זה היא להדגים את השימוש המתאים של microarrays חלבון הפונקציונלי כדי לזהות אינטראקציות חלבון-חלבון. יישום זה מאפשר הניתוח ביוכימי המקביל תפוקה גבוהה של פעילות חלבון מחייב שימוש אנליטי נקי במיוחד (חלבון) של עניין. C-מסוף (carboxy מסוף) מתויג חלבון פיתיון V5-היתוך של העניין מופק מפלסמיד גבוה עותק בזן שמרים מותאמים לטיהור חלבונים. תיוג C-המסוף מבטיח כי החלבון באורך מלא תורגם. החלבון השתמש במחקר זה הוא קינאז חלבון היתוך Tda1-V5, אשר מטוהר באמצעות שרף זיקת ניקל באמצעות תג His6X. CONSTRUC היתוך Tda1-V5לא מטוהר באמצעות elution הסידורי באמצעות שיפוע imidazole לelute שבריר ביותר המועשר לשימוש בassay.

<table><tbody><tr><td>Petri Dishes</td><td>VWR</td><td>NC-10747</td><td>or comparable</td></tr><tr><td>Bacto yeast extract</td><td>Difco</td><td>0217-17</td><td></td></tr><tr><td>Bacto peptone</td><td>Difco</td><td>0118-17</td><td></td></tr><tr><td>Bacto agar</td><td>Difco</td><td>0140-01</td><td></td></tr><tr><td>Dextrose</td><td>Sigma</td><td>D9434</td><td></td></tr><tr><td>Raffinose</td><td>Sigma</td><td>R0514</td><td></td></tr><tr><td>Galactose</td><td>Sigma</td><td>G0750</td><td></td></tr><tr><td>Sc-Ura drop out media</td><td>MP Bio</td><td>114410622</td><td></td></tr><tr><td>Small Culture tubes</td><td>VWR/Fisher</td><td></td><td></td></tr><tr><td>Large Erlenmeyer Flask</td><td>VWR/Fisher</td><td></td><td></td></tr><tr><td>Centrifuge JA-10 rotor</td><td></td><td></td><td></td></tr><tr><td>Centrifuge tubes (500 ml)</td><td></td><td></td><td></td></tr><tr><td>Falcon tube (50 ml)</td><td>VWR/Fisher</td><td>21008-951</td><td></td></tr><tr><td>PBS Tablets</td><td>Sigma</td><td>P4417</td><td></td></tr><tr><td>FastPrep Tubes (2 ml screw cap tube)</td><td></td><td></td><td></td></tr><tr><td>FastPrep Machine</td><td>MP Biomedicals</td><td>116004500</td><td></td></tr><tr><td>Zircon Beads</td><td>BioSpec</td><td>11079105</td><td></td></tr><tr><td>Triton X100</td><td>Sigma</td><td>P4417</td><td></td></tr><tr><td>DTT</td><td>Sigma</td><td>D9779</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266&amp;nbsp;</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S3014&amp;nbsp;</td><td></td></tr><tr><td>Imidazole (pH = 7.4)</td><td>Sigma</td><td>I5513&amp;nbsp;</td><td></td></tr><tr><td>PMSF</td><td>Sigma</td><td>93482</td><td></td></tr><tr><td>Complete Inhibitor Cocktail (EDTA Free)</td><td>Roche</td><td>11873580001</td><td></td></tr><tr><td>Phosphatase inhibitor cocktail 1</td><td>sigma</td><td>P2850</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A2153&amp;nbsp;</td><td></td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P1379&amp;nbsp;</td><td></td></tr><tr><td>ATP</td><td>Sigma</td><td>A1852&amp;nbsp;</td><td></td></tr><tr><td>Shaking Incubator (30 &amp;deg;C)</td><td></td><td></td><td></td></tr><tr><td>Nickel Affinity Resin</td><td>Life Technologies</td><td>R901-01</td><td></td></tr><tr><td>G25 column</td><td>GE life sciences</td><td>27-5325-01&amp;nbsp;</td><td></td></tr><tr><td>Nutator</td><td>VWR/Fisher</td><td></td><td></td></tr><tr><td>SDS-PAGE Gel (NuPage)</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Coomassie Blue Stain</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Monoclonal V5 Antibody</td><td>Life Technologies</td><td>R960-25</td><td></td></tr><tr><td>Alexa647 Tagged monoclonal V5 Antibody</td><td>Life Technologies</td><td>451098</td><td></td></tr><tr><td>Protein Microarrays Kit</td><td>Life Technologies</td><td>PAH0525013</td><td></td></tr><tr><td>Genepix Scanner</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Lifter Slips</td><td>Thomas Scientific</td><td></td><td>http://www.thomassci.com/Supplies/Microscope-Cover-Glass/_/LIFTER-SLIPS/</td></tr><tr><td>Lysis Buffer: 0.1% Triton X-100, 0.5 mM DTT,&lt;br/&gt; 2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 500 mM NaCl, 50 mM imidazole (pH 7.4), Complete protease inhibitor cocktail &amp;ndash; EDTA-free, Phosphatase inhibitor Cocktail 1, 1 mM PMSF&amp;nbsp; (add just prior to use)</td><td></td><td></td><td>Add the reagents to 1x PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride; pH 7.4) to obtain the desired volume of lysis solution</td></tr><tr><td>Blocking Buffer: 1% bovine serum albumin (BSA), 0.1% Tween-20</td><td></td><td></td><td>Add the reagents to 1x PBS to obtain the desired volume of blocking buffer</td></tr><tr><td>Probe Buffer: 2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 0.5 mM DTT, 0.05% Triton X-100, 50 mM NaCl, 500 &amp;mu;M ATP (for kinases), 1% BSA</td><td></td><td></td><td>Add the reagent to 1x PBS to obtain desired volume of probe buffer</td></tr><tr><td>Wash Buffer: 0.1% Triton X-100, 500 mM NaCl, 0.5 mM DTT, 50 mM imidazole (pH 7.4),2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td>Add the reagent to 1x PBS to obtain desired volume of probe buffer</td></tr><tr><td>Elution Buffer: 0.1% Triton X-100, 0.5 mM DTT, 2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 500 mM NaCl, 50 &amp;ndash; 500 mM imidazole (pH 7.4), Complete protease inhibitor cocktail &amp;ndash; EDTA-free, Phosphatase inhibitor Cocktail 1, 1 mM PMSF (add just prior to use)</td><td></td><td></td><td>Before you begin, it is important to note the concentration gradient of imidazole (50 &amp;ndash; 500 mM) and to create separate tubes for each concentration (it is advisable to create the interval using 50 mM increments). Add the reagent to 1x PBS to obtain desired volume of probe buffer</td></tr><tr><td>3x YEP +6% galactose: Dissolve 30 g of yeast extract and&amp;nbsp; 60 g of peptone in 700 ml of H2O, and autoclave. Add 300 ml of filter sterilized 20% galactose (wt/vol)</td><td></td><td></td><td>&amp;nbsp;Galactose should not be autoclaved</td></tr></tbody></table>

probing high-density functional protein microarrays to detect protein-protein interactions

microarrays חלבון הפונקציונלי בצפיפות גבוהה המכיל ~ 4,200 חלבוני שמרי רקומביננטי נבחנים לאינטראקציות חלבון-חלבון קינאז באמצעות חלבון היתוך הזיקה מטוהרת השמרים קינאז המכיל תג V5-epitope לקריאה החוצה. קינאז המטוהר מתקבל באמצעות התרבות של זן שמרים מותאמים לייצור חלבון גבוה עותק מחסה פלסמיד המכיל היתוך קינאז-V5 לבנות תחת אמרגן מושרה GAL. השמרים הוא גדלו בתקשורת כובל עם מקור פחמן ניטראלי עבור 6 שעות אחרי אינדוקציה עם גלקטוז 2%. בשלב הבא, התרבות שנקטפו וקינאז הוא מטוהר באמצעות טכניקות chromatographic זיקה סטנדרטית להשיג קינאז חלבון נקי במיוחד לשימוש בassay. קינאז המטוהר מדולל עם חיץ קינאז לטווח מתאים עבור assay וmicroarrays החלבון חסום לפני הכלאה עם microarray החלבון. לאחר ההכלאה, המערכים מששו עם antib V5 חד שבטיody לזהות חלבונים מחויבים לחלבון קינאז-V5. לבסוף, המערכים נסרקים באמצעות סורק microarray סטנדרטי, והנתונים מופק לניתוח אינפורמטיקה במורד הזרם 1,2 לקבוע אמון גבוה שנקבע של אינטראקציות חלבון לאימות במורד הזרם in vivo.

פעילות האינטראקציה בין חלבונים נצפתה באמצעות קורא שבבים סטנדרטי על מנת להעריך את היתוך החלבון kinase Tda1-V5 לבנות כחלבון פיתיון נגד microarray חלבון פונקציונלי שמרים המכיל כ -4,200 ס הייחודי חלבוני GST-היתוך cerevisiae. חקירה נוספת עם תוכנת GenePix גילתה שפע של אירועים מחייבים ש?...

Watch this Scientific Journal Video about Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions at JoVE.com

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

באמצעות מערכי חלבון המכיל כמעט את כל ס proteome cerevisiae הוא חקר לחקירה משוחדת מהירה של אלפי אינטראקציות חלבון-חלבון במקביל. שיטה זו יכולה להיות מנוצלת למולקולת חלבון קטן, שינוי posttranslational, ומבחנים אחרים בתפוקה גבוהה.

חיטוט Microarrays חלבון פונקציונלי בצפיפות גבוהה לזיהוי חלבונים אינטראקציות

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

probing-high-density-functional-protein-microarrays-to-detect-protein

Research

JoVE Journal

Biology

8.0K Views.  Stanford University.  באמצעות מערכי חלבון המכיל כמעט את כל ס proteome cerevisiae הוא חקר לחקירה משוחדת מהירה של אלפי אינטראקציות חלבון-חלבון במקביל. שיטה זו יכולה להיות מנוצלת למולקולת חלבון קטן, שינוי posttranslational, ומבחנים אחרים בתפוקה גבוהה. 

Video: Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

פתרון מתחמי פרוטאין טהור זיקה לפי דף Native הכחול פרופיל מתאם חלבון

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Biochemistry

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

חלבון חוץ-תאית הטכנולוגיה Microarray לגילוי תפוקה גבוהה של אינטראקציות קולטן-ליגנד זיקה נמוכה

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

גישה השוואתית כדי לאפיין את הנוף של אינטראקציות בין חלבונים מאכסן לפתוגן

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Immunology and Infection

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

קוונפיקציה של אינטראקציה חלבון ברשת דינמיקה באמצעות ריבוב ושות-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

In-vivo גילוי של חלבונים אינטראקציות על מיקרו-בדוגמת משטחים

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

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.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

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 ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

זיהוי של שותפי אינטראקציה חלבון בתאי יונקים באמצעות SILAC-immunoprecipitation פרוטאומיקה כמותי

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

זיהוי אתרי אינטראקציה בין חלבונים באמצעות פפטיד מערכים

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

הקרנת הגנום כל חלבונים אינטראקציה ידי חלבון-בר השלמה Assay (PCA) בתאים חיים

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Protein-protein Interfaces

Many proteins form complexes to carry out their functions, making protein-protein interactions (PPIs) essential for an organism&#39;s survival. Most PPIs are stabilized by numerous weak noncovalent chemical forces. The physical shape of the interfaces determines the way two proteins interact. Many globular proteins have closely-matching shapes on their surfaces, which form a large number of weak bonds. Additionally, many PPIs occur between two helices or between a surface cleft and a polypeptide chain or string.
Various computational and biochemical methods are used to study protein interfaces. Laboratory methods, such as affinity purification, mass spectrometry, and protein microarrays, can be used to identify new interactions. Co-immunoprecipitation of proteins and yeast two-hybrid screening are widely used to provide evidence on whether two proteins interact in vitro.&#160; Computer programs can predict PPIs based on similar interactions found in other proteins by comparing protein sequences and three-dimensional structures. Other computational approaches, such as phylogenetic profiling, predict PPIs based on the coevolution of binding partners. Additionally, gene fusion analysis is used to predict interaction partners by finding protein pairs that are fused in the genome of other organisms.&#160;
Proteins typically interact with multiple partners either at the same or different times, and they may contain more than one interaction interface.&#160; Many proteins form large complexes that perform specific functions that can only be carried out by the complete complex. In some cases, these protein interactions are regulated; that is, a protein may interact with different partners based on cellular needs. Further computational and statistical analyses sort such interactions into networks, which are curated in online interactome databases. These searchable databases enable users to study specific protein interactions, as well as design drugs that can enhance or disrupt interactions at the interface.

Protein Complexes and Protein-Protein Interactions

ממשקי חלבון-חלבון

Many proteins form complexes to carry out their functions, making protein-protein interactions (PPIs) essential for an organism&#39;s survival. Most PPIs ...