משרד ההגנה (פרס הרעיון W81XWH-07-0406) - כדי Ulus אטסוי NIH RO1 A1080870 - כדי Ulus אטסוי NIH R21 A1079341 - כדי Ulus אטסוי אוניברסיטת מיזורי קרנות מוסדיות - כדי Ulus אטסוי

<ol>
	<li>Calaluce, R., Gubin, M. M., Davis, J. W., Magee, J. D., Chen, J., Kuwano, Y., Gorospe, M., Atasoy, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNA+binding+protein+HuR+differentially+regulates+unique+subsets+of+mRNAs+in+estrogen+receptor+negative+and+estrogen+receptor+positive+breast+cancer.">The RNA binding protein HuR differentially regulates unique subsets of mRNAs in estrogen receptor negative and estrogen receptor positive breast cancer.</a> BMC Cancer. 10, 126-140 (2010).</li><li>Gubin, M. M., Calaluce, R., Davis, J. W., Magee, J. D., Strouse, C. S., Shaw, D. P., Ma, L., Brown, A., Hoffman, T., Rold, T. L., Ulus Atasoy, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+the+RNA+binding+protein+HuR+impairs+tumor+growth+in+triple+negative+breast+cancer+associated+with+deficient+angiogenesis.">Overexpression of the RNA binding protein HuR impairs tumor growth in triple negative breast cancer associated with deficient angiogenesis.</a> Cell Cycle. 9, 3337-3346 (2010).</li><li>Keene, J. D., Komisarow, J. M., Friedersdorf, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RIP-Chip:+the+isolation+and+identification+of+mRNAs,+microRNAs+and+protein+components+of+ribonucleoprotein+complexes+from+cell+extracts.">RIP-Chip: the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts.</a> Nature Protocols. 1, 302-307 (2006).</li><li>Tenenbaum, S. A., Lager, P. J., Carson, C. C., Keene, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribonomics:+identifying+mRNA+subsets+in+mRNP+complexes+using+antibodies+to+RNA-binding+proteins+and+genomic+arrays.">Ribonomics: identifying mRNA subsets in mRNP complexes using antibodies to RNA-binding proteins and genomic arrays.</a> Methods. 26, 191-198 (2002).</li><li>Tenenbaum, S. A., Carson, C. C., Atasoy, U., Keene, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+regulatory+analysis+using+en+masse+nuclear+run-ons+and+ribonomic+profiling+with+autoimmune+sera.">Genome-wide regulatory analysis using en masse nuclear run-ons and ribonomic profiling with autoimmune sera.</a> Gene. 317, 79-87 (2003).</li><li>Tenenbaum, S. A., Carson, C. C., Lager, P. J., Keene, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+mRNA+subsets+in+messenger+ribonucleoprotein+complexes+by+using+cDNA+arrays.">Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays.</a> Proc. Natl. Acad. Sci. U.S.A. 97, 14085-14090 (2000).</li><li>Baroni, T. E., Chittur, S. V., George, A. D., Tennebaum, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+RIP-Chip+Analysis.">Advances in RIP-Chip Analysis.</a> Methods in Molecular Biology. 419, 93-108 (2008).</li><li>de Silanes Lopez, I., Zhan, M., Lal, A., Yang, X., Gorospe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+target+RNA+motif+for+RNA-binding+protein+HuR.">Identification of a target RNA motif for RNA-binding protein HuR.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 2987-2992 (2004).</li><li>de Silanes Lopez, I., Lal, A., Gorospe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HuR:+post-transcriptional+paths+to+malignancy.">HuR: post-transcriptional paths to malignancy.</a> RNA Biol. 2, 11-13 (2005).</li><li>Grigull, J., Mnaimneh, S., Pootoolal, J., Robinson, M. D., Hughes, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+mRNA+stability+using+transcription+inhibitors+and+microarrays+reveals+posttranscriptional+control+of+ribosome+biogenesis+factors.">Genome-wide analysis of mRNA stability using transcription inhibitors and microarrays reveals posttranscriptional control of ribosome biogenesis factors.</a> Mol. Cell Biol. 24, 5534-5547 (2004).</li><li>Hieronymus, H., Silver, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+RNA-protein+interactions+illustrates+specificity+of+the+mRNA+export+machinery.">Genome-wide analysis of RNA-protein interactions illustrates specificity of the mRNA export machinery.</a> Nat. Genet. 33, 155-161 (2003).</li><li>Hieronymus, H., Yu, M. C., Silver, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+mRNA+surveillance+is+coupled+to+mRNA+export.">Genome-wide mRNA surveillance is coupled to mRNA export.</a> Genes Dev. 18, 2652-2662 (2004).</li><li>Mukherjee, N., Corcoran, D. L., Nusbaum, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+Regulatory+Mapping+Indicates+that+the+RNA-Binding+Protein+HuR+Couples+Pre-mRNA+Processing+and+mRNA+Stability.">Integrative Regulatory Mapping Indicates that the RNA-Binding Protein HuR Couples Pre-mRNA Processing and mRNA Stability.</a> Molecular Cell. 43, 1-13 (2011).</li></ol>

החוקרים מצהירים כי אין להם אינטרסים מתחרים.

בשל האופי של ניסוי, אופטימיזציה וניסיון זה יהיו הדרכים מובטחות רק כדי לרכוש את התוצאות המצופות בהצלחה. בשלבים רבים של הליך זה, טמפרטורה וטיפול יעיל של חומרים הכימיים ומוצרים הם חשובים וקריטיים. תכנון וביצוע של טכניקה נכון יעזרו להבטיח כי הניסוי בוצע במסגרת זמן מתאימה בטמפרטורה האופטימלית המומלצת. נושא מרכזי בניסויי בידוד RNA הוא הרגישות של RNAs לשפלה ידי RNases. כל ריאגנטים צריכים להיות RNase חופשי ומאוחסן או משמש במכולות RNase חינם. זה הוא שלב קריטי בהבטחת השלמות של מדגם mRNA שלך. גם כאשר הניסוי מבוצע כראוי, עם זאת, התוצאה הרצויה לא יכולה להיות מושגת בשל האופי של האינטראקציה בין RBP וmRNAs היעד שלה. בעיה פוטנציאלית אחת היא שיש נמוכה או אפילו אין אות מרנ&quot;א מבודד על ידי RIP שבב.למרות שעשוי להיות אות מ-RNA המוחלט, זה עשוי להיות תוצאה של חלבון מחייב לקוי שנגרר על ידי החרוזים. הצעד הראשון הוא פתרון הבעיות כדי לאשר שlysate הסלולרי בשימוש יש ביטוי נאות RBP הספציפי. עם האישור, חלבון יכול להיות מבודד לאחר NT2 השטיפה הסופית וresuspended במאגר Laemmli או אחר חיץ denaturing מתאים ומחומם על 95 מעלות צלזיוס למשך 5 דקות. ניתוח הכתם מערבי ניתן להשתמש בדגימות אלה בתיאום עם lysate קלט כמו גם בקרות שליליות כדי להבטיח מספיק למשוך למטה של ​​חלבון קשור. יתר על כן, בגלל lysing התא נדרש כדי לגשת לרכיבים אלה, הפוטנציאל לאינטראקציות בלתי תקינות ובלתי רצויות בין חלבונים וmRNA בדרך מופרדים עשוי להיות מוצג. אינטראקציות אלה עלולות להיקשר ו&quot; לספוג &quot;mRNAs היעד שלך או החלבונים מחייבים דרך אינטראקציות לא ספציפיות. בנוסף, חלבונים אלו בשיתוף שונהnditions יכול לקפל בוריאציות שונות, והמוטיבים המחייבים שלהם עשויים להיות נגישים לmRNAs היעד שלהם, ומונעים האינטראקציות ביניהם. שני אלה מחזקים את החשיבות של עבודה ביעילות, כמו גם ניצול האופטימלי הטמפרטורות מפורטים להגביל אינטראקציות בלתי רצויות אלה. בנוסף, אופטימיזציה של שטיפת תנאים לכל חלבון המטרה ספציפי תהיה קריטית למקסם את הטוהר של האינטראקציה. תנאים מחמירים יותר להדחה עשויים להיות נחוצים. לדוגמה, החיץ לשטוף ניתן להשלים עם SDS או כמות מתאימה של אוריאה להפחית אינטראקציות ורקע לא ספציפיות באות הפלט שלך. זה יהיה תלוי לחלוטין בRBP היעד של הנסיין, כמו גם את ה-mRNA היעד בתנאים הפיסיולוגיים הייחודיים שלהם. תנאים מסוימים לא יהיו מתאימים לכלים מסוימים mRNA ניתוח, שיצוינו בהכנת דגימות. לבסוף, למרות RIP הוא מוצלח בהעשרה של רנ&quot;אאינטראקציות RBP, בעיה מוכרת היטב עם שיטת RIP (שבב) היא חוסר היכולת לזהות את התחומים המחייבים הספציפיים של RBP על יעדי mRNA החולפים. כמה טכניקות צלב מקשר-יכולים לשמש אחרי RIP לבודד יעדי רצף ייחודיים, עם זאת, השימוש בקרינת UV בגלים קצרים נוטה להוביל לניזק חומצות גרעין. שיטה חדשה המכונית PAR-CLIP, או crosslinking ribonucleoside photoactivatable וimmuoprecipitation, מעסיקה ארוך UV-גל לשלב thiouridine לרנ&quot;א המתהווה המאפשר זיהוי של אתרי קישור ייחודיים מאינטראקציות RNA הוא יציבות וחולפות. בסך הכל, שבב RIP הוקם ככלי מצוין המשמש לבודד ולבחון את יחסי גומלין בין חלבונים המחייבים RNA ויעדי mRNA שלהם על ידי הקבוצה שלנו, כמו גם רבות קבוצות מחקר אחרות. למרות רגיש בטבע ובפועל, ביצוע נכון של הליך זה יניב את הבידוד של קומפלקסי RNP אלה, שעד לא מזמן, היה inaccessible לגילוי וניתוח.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגים </td><td> תגובות </td></tr><tr><td> 1 ז dithiothreitol </td><td> דיג </td><td> BP172-5 </td><td> DTT </td></tr><tr><td> 1 DNase </td><td> Ambion </td><td> 2235 </td><td> RNase חופשי </td></tr><tr><td> Ethylendiamine חומצת Tetraccetic </td><td> דיג </td><td> BP118-500 </td><td> EDTA </td></tr><tr><td> גליקוגן </td><td> Ambion </td><td> 9516 </td><td></td></tr><tr><td> 1 HEPES </td><td> סיגמא </td><td> H3375-100G </td><td> pH 7.0 </td></tr><tr><td> Igepal Nonidet P-40 </td><td> USB </td><td> 78641 </td><td> NP40 </td></tr><tr><td> 1 ז KCl </td><td> דיג </td><td> BP366-500 </td><td></td></tr><tr><td> 1 ז MgCl 2 </td><td> דיג </td><td> BP214-500 </td><td></td></tr><tr><td> NT2 מאגר </td><td> * ראה להלן </td><td></td><td></td></tr><tr><td> מאגר תמוגה Polysome </td><td> * ראה להלן </td><td></td><td></td></tr><tr><td> טבליות קוקטייל מעכב פרוטאז </td><td> רוש </td><td> 11873580001 </td><td></td></tr><tr><td> חלבון חרוזי Sepharose </td><td> סיגמא </td><td> P3391 </td><td></td></tr><tr><td> Proteinase K </td><td> דיג </td><td> BP1700-100 </td><td></td></tr><tr><td> RNase אאוט RNase המעכב </td><td> Invitrogen </td><td> 10777-019 </td><td> 40 U / μl </td></tr><tr><td> 1 ז NaCl </td><td> דיג </td><td> BP358-212 </td><td></td></tr><tr><td> סולפט נתרן dodecyl </td><td> דיג </td><td> BP166-500 </td><td> SDS </td></tr><tr><td> 1 ז טריס-HCl </td><td> דיג </td><td&gt; BP153-500 </td><td> pH 7.4 </td></tr><tr><td> Trizol </td><td> Invitrogen </td><td> 15596-026 </td><td></td></tr><tr><td> מכלולי Ribonucleoside Vanadyl </td><td> מעבדות ניו אינגלנד </td><td> S1402S </td><td> VRC </td></tr></tbody></table> <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> בירור מגיב </td></tr><tr><td> הכן ריאגנטים בRNase / DNase חינם, כלי הזכוכית DEPC טופלה </td></tr><tr><td> מאגר תמוגה Polysome </td></tr><tr><td> 100 המ&quot;מימ KCl </td></tr><tr><td> 5 המ&quot;מ MgCl 2 </td></tr><tr><td> 10 HEPES מ&quot;מ (pH 7.0) </td></tr><tr><td> 0.5% NP40 </td></tr><tr><td> 1 המ&quot;מ DTT </td></tr><tr><td> 100 יחידות / מיליליטר RNase אאוט </td></tr><tr><td> 400 מיקרומטר VRC </td></tr><tr><td> לוח קוקטייל מעכב פרוטאז </td></tr><tr><td> 5 מ&quot;ל של חיץ תמוגה Polysome </td></tr><tr><td> הוסף 50 μl של 1 HEPES M (pH 7.0) </td></tr><tr><td> 500 μl של ז KCL 1 </td></tr><tr><td> 25 μl של ז MgCl 1 2 </td></tr><tr><td> 25 μl של NP40 </td></tr><tr><td> 4.7 המ&quot;ל RNase-DNase ללא H 2 O </td></tr><tr><td> 50 μl של M-DTT 1 </td></tr><tr><td> 12.5 μl של 100 U / המ&quot;ל RNase אאוט </td></tr><tr><td> קוקטייל 200 μl פרוטאז מעכב (מומס על פי יצרן) </td></tr><tr><td> 10 μl 200 המ&quot;מ VRC (בזמן שימוש) </td></tr><tr><td> NT2 מאגר </td></tr><tr><td> 50 mM טריס-HCl (pH 7.4) </td></tr><tr><td> 150 המ&quot;מ NaCl </td></tr><tr><td> 1 המ&quot;מ MgCl 2 </td></tr><tr><td> 0.05% NP40 </td></tr><tr><td> 1 ליטר של NT2 מאגר </td></tr><tr><td> 50 המ&quot;ל טריס (pH 7.4) </td></tr><tr><td> 30 מ&quot;ל 5 M NaCl </td></tr><tr><td> 1 מ&quot;ל 1 ז MgCl <sub&gt; 2 </td></tr><tr><td> 500 NP40 μl </td></tr><tr><td> 820 המ&quot;ל RNase-DNase ללא H 2 O </td></tr></tbody></table>

method for the isolation and identification of mrnas, micrornas and protein components of ribonucleoprotein complexes from cell extracts using rip-chip

כתוצאה מההתפתחות של רצף תפוקה גבוהה וניתוח microarray יעיל, ניתוח ביטוי גנים העולמי הפך צורה קלה וזמינה של איסוף נתונים. במחקר רב ומודלים למחלות עם זאת, רמות המצב יציבות של mRNA גן היעד לא תמיד לתאם ישירות עם רמות חלבון המצב יציבות. רגולציה של גנים לאחר השעתוק היא הסבר סביר של הסטייה בין שתיים. מונע על ידי הקשירה של חלבונים מחייבים RNA (RBP), רגולציה שלאחר השעתוק משפיעה לוקליזציה-mRNA, יציבות ותרגום על ידי יצירת Ribonucleoprotein (RNP) מורכבת עם mRNAs היעד. זיהוי מטרות de mRNA נובו הידועות אלה מתמציות סלולריות במתחם RNP הוא מרכזי להבנת מנגנונים ופונקציות של RBP וההשפעה שלהם על תוצאת פלט חלבון. פרוטוקול זה מתאר שיטה כינתה RNP immunoprecipitation-microarray (RIP-Chip), המאפשרת לזיהוי שלmRNAs pecific קשור במתחם ribonucleoprotein, תחת תנאי ניסוי משתנה, יחד עם אפשרויות לייעול נוסף לניסוי לחוקר הבודד. בכלי ניסיוני חשוב זה, חוקרים יכולים לחקור את המנגנונים המורכבים הקשורים לרגולציה של גנים שלאחר שעתוק, כמו גם אינטראקציות ribonucleoprotein אחרות.

Watch this Scientific Journal Video about Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip at JoV

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

צעד אחר צעד לפרוטוקול בידוד וזיהוי RNA מתחמים הקשורים באמצעות RIP שבב.

שיטה לבידוד וזיהוי mRNAs, רנ&quot;א ורכיבי חלבון של מכלולי Ribonucleoprotein מתמציות סלולריות באמצעות שבב RIP

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

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27.4K Views.  University of Missouri. צעד אחר צעד לפרוטוקול בידוד וזיהוי RNA מתחמים הקשורים באמצעות RIP שבב.

Video: Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)1, and the negative staining has been performed by Wu et al. (2007).2

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.

טיהור ויזואליזציה של שפעת מכלולים Ribonucleoprotein ויראלי

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

זיהוי של קומפלקסים חלבונים עם proteomics כמותיים cerevisiae ס

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

בידוד יציב Multi-חלבון על ידי תסביכים In vivo מבוקר נייד חוצה קישור חיסונית מגנטי כרומטוגרפיה זיקה

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

זיהוי ואפיון של חלבונים באמצעות glycosylation ספציפי אנדו ו exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

זיהוי של קומפלקסי חלבונים ב<em&gt; חיידקי Escherichia</em&gt; באמצעות טיהור זיקת פפטיד סדרתית בשילוב עם טנדם המוני ספקטרומטריית

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

זיהוי לאחר translational השינויים של קומפלקסי חלבוני צמח

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

בידוד של דם המופק מכולים multipotent מבשרים משרירי שלד אדם

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

מיפוי מבוסס AFM של תכונות אלסטיות של קירות תא: ברקמות, נייד, והחלטות subcellular

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

נוהל כדי להעריך את היעילות של flocculants להרחקת מפוזרי חלקיקים מן תמציות צמחים

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...

Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes

Sampling the phloem of higher plants is often laborious and significantly dependent on the plant species. However, proteome studies under denaturing conditions could be achieved in different plant species. Native protein:protein and protein:nucleic acid complexes from phloem samples have as yet scarcely been analyzed, although they might play important roles in maintenance of this specialized compartment or in long-distance signaling. Large molecular assemblies can be isolated using a blue native gel electrophoresis (BN-PAGE). Their protein components can be separated by a subsequent sodium dodecyl sulfate PAGE (SDS-PAGE). However, proteins with similar molecular weights co-migrate, what can hinder protein identification by mass spectrometry. Combining BN-PAGE with two different denaturing gel electrophoresis steps, namely Tris-Tricine-urea and SDS-PAGE, enables the additional separation of proteins according to their hydrophilicity/hydrophobicity and thus increases resolution and the success of protein identification. It even allows distinguishing proteins that only differ in their posttranslational modifications. In addition, blue native northern blotting can be applied to identify the RNA components in macromolecular complexes. We show that our protocol is suitable to unravel the protein and RNA components of native protein:protein and ribonucleoprotein (RNP) complexes occurring in phloem samples. Combining a blue native PAGE with two different denaturing PAGE steps can help to separate different kinds of large protein complexes, and also enables an increased identification rate of their components by mass spectrometry. Furthermore, the protocol is robust enough to simultaneously detect potentially bound nucleic acids within single protein complexes.

Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes

Here we present a protocol to analyze the protein composition of large native protein:protein and protein:nucleic acid complexes from oilseed rape (B. napus) phloem exudate using a 3D polyacrylamide gel electrophoresis (PAGE) approach combining blue native (BN) with two denaturing PAGEs followed by mass spectrometric identification.

שיפה Sap דגימה של כרוב napus עבור תלת-ממד-דף של חלבונים, קומפלקסים Ribonucleoprotein

Sampling the phloem of higher plants is often laborious and significantly dependent on the plant species. However, proteome studies under denaturing ...