The authors are grateful for the support and advice of Nikolaus Grigorieff. We thank Anna Loveland, Axel Brilot, Chen Xu, and Mike Rigney for helpful discussions and EM guidance. This work was funded by the National Science Foundation, Award No. 1157892. The Brandeis EM facility is supported by National Institutes of Health grant P01 GM62580.

<ol>
	<li>Gavin, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+organization+of+the+yeast+proteome+by+systematic+analysis+of+protein+complexes.">Functional organization of the yeast proteome by systematic analysis of protein complexes.</a> Nature. 415 (6868), 141-147 (2002).</li><li>Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M., Seraphin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+generic+protein+purification+method+for+protein+complex+characterization+and+proteome+exploration.">A generic protein purification method for protein complex characterization and proteome exploration.</a> Nature Biotechnology. 17 (10), 1030-1032 (1999).</li><li>Puig, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tandem+affinity+purification+(TAP)+method:+A+general+procedure+of+protein+complex+purification.">The tandem affinity purification (TAP) method: A general procedure of protein complex purification.</a> Methods. 24 (3), 218-229 (2001).</li><li>Vaillancourt, P., Zheng, C. F., Hoang, D. Q., Breister, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+purification+of+recombinant+proteins+fused+to+calmodulin+or+to+calmodulin-binding+peptides.">Affinity purification of recombinant proteins fused to calmodulin or to calmodulin-binding peptides.</a> Methods Enzymol. 326, 340-362 (2000).</li><li>Braisted, A. C., Wells, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimizing+a+binding+domain+from+protein+A.">Minimizing a binding domain from protein A.</a> Proc Natl Acad Sci U S A. 93 (12), 5688-5692 (1996).</li><li>Kapust, R. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+etch+virus+protease:+mechanism+of+autolysis+and+rational+design+of+stable+mutants+with+wild-type+catalytic+proficiency.">Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency.</a> Protein Eng. 14 (12), 993-1000 (2001).</li><li>Nallamsetty, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+site-specific+processing+of+fusion+proteins+by+tobacco+vein+mottling+virus+protease+in+vivo+and+in+vitro.">Efficient site-specific processing of fusion proteins by tobacco vein mottling virus protease in vivo and in vitro.</a> Protein Expr Purif. 38 (1), 108-115 (2004).</li><li>Ghaemmaghami, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+protein+expression+in+yeast.">Global analysis of protein expression in yeast.</a> Nature. 425 (6959), 737-741 (2003).</li><li>Howson, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction,+verification+and+experimental+use+of+two+epitope-tagged+collections+of+budding+yeast+strains.">Construction, verification and experimental use of two epitope-tagged collections of budding yeast strains.</a> Comp Funct Genomics. 6 (1-2), 2-16 (2005).</li><li>Cox, D. M., Du, M., Guo, X., Siu, K. W., McDermott, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tandem+affinity+purification+of+protein+complexes+from+mammalian+cells.">Tandem affinity purification of protein complexes from mammalian cells.</a> Biotechniques. 33 (2), 267-268 (2002).</li><li>Gully, D., Moinier, D., Loiseau, L., Bouveret, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+partners+of+acyl+carrier+protein+detected+in+Escherichia+coli+by+tandem+affinity+purification.">New partners of acyl carrier protein detected in Escherichia coli by tandem affinity purification.</a> FEBS Lett. 548 (1-3), 90-96 (2003).</li><li>Tharun, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+analysis+of+the+decapping+activator+Lsm1p-7p-Pat1p+complex+from+yeast.">Purification and analysis of the decapping activator Lsm1p-7p-Pat1p complex from yeast.</a> Methods Enzymol. 448, 41-55 (2008).</li><li>Xu, X., Song, Y., Li, Y., Chang, J., Zhang, H., An, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tandem+affinity+purification+method:+an+efficient+system+for+protein+complex+purification+and+protein+interaction+identification.">The tandem affinity purification method: an efficient system for protein complex purification and protein interaction identification.</a> Protein Expr Purif. 72 (2), 149-156 (2010).</li><li>Cheng, Y., Grigorieff, N., Penczek, P. A., Walz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Primer+to+Single-Particle+Cryo-Electron+Microscopy.">A Primer to Single-Particle Cryo-Electron Microscopy.</a> Cell. 161 (3), 438-449 (2015).</li><li>Gottschalk, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+biochemical+and+genetic+analysis+of+the+yeast+U1+snRNP+reveals+five+novel+proteins.">A comprehensive biochemical and genetic analysis of the yeast U1 snRNP reveals five novel proteins.</a> RNA. 4 (4), 374-393 (1998).</li><li>Neubauer, G., Gottschalk, A., Fabrizio, P., Seraphin, B., Luhrmann, R., Mann, 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+the+proteins+of+the+yeast+U1+small+nuclear+ribonucleoprotein+complex+by+mass+spectrometry.">Identification of the proteins of the yeast U1 small nuclear ribonucleoprotein complex by mass spectrometry.</a> Proc Natl Acad Sci U S A. 94 (2), 385-390 (1997).</li><li>Lucast, L. J., Batey, R. T., Doudna, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+purification+of+a+stable+form+of+recombinant+tobacco+etch+virus+protease.">Large-scale purification of a stable form of recombinant tobacco etch virus protease.</a> Biotechniques. 30 (3), 544-546 (2001).</li></ol>

The authors have nothing to disclose.

השיטה TAP מנצל שני תגים לאזן צורך חזק וסלקטיבי מחייב שרף זיקה עם רצון לשמור כולה במזג אוויר פתרון פיזיולוגי. איזון זה משמש כדי לשמר אינטראקציה יציבה (ים) של החלבון מתויג עם אינטראקצית גורם (ים) לאפיון שלאחר טיהור. בנוסף, TAP הפרט מתויג ORFs זמין ממקור מסחרי, כך שאף אחד יכול ל?...

תהליכים תאיים גדולים רבים מבוצעים על ידי קומפלקסי חלבונים גדולים החלבון-RNA 1. צוואר בקבוק משמעותי לעריכת מחקרי biophysical והמבניים של קומפלקסים כאלה הוא מקבל אותם באיכות מתאימה (כלומר, הומוגניות) ותמורת ריכוז מתאים. בידוד קומפלקס ממקור ילידים יש יתרונות רבים, כולל שמירת שינויים שלאחר תעתיק ו / או translational רלוונטיים של יחידות משנה וביטוח הרכבה מורכבת ראויה. עם זאת, מתחמי הסלולר גדולים הם בדרך כלל נוכח בתא לעבר מספר נמוך עותק ואת הטיהור חייבת להיות יעילה להתרחש בתנאים פיסיולוגיים ליד מנת לשמור על שלמות מורכבת נשמרה. טיהור קומפלקס ממקור איקריוטיים מאתגרת במיוחד ולא יכול להיות כלכלי מונע. לכן, אסטרטגיות או שיטות שאינן יעילים להניב מורכבים הומוגנית הן מאוד רצויות. אסטרטגיה כי כברמוצלח לטיהור מתחמי יליד מתאי איקריוטיים לאפיון הראשוני שלהם הוא טיהור זיקת טנדם 2,3 השיטה (TAP). שיטת TAP בתחילה תוכננה כדי לטהר חלבון יליד מן שמרי ניצנים (cerevisiae ס) בקומפלקס עם אינטראקצית גורם (ים) 2. שיטת TAP מנוצלת שני תגים, כל תג התמזג במקביל לאותו רצף גן מקודדי חלבונים. התגים נבחרו כדי לאזן צורך חזק וסלקטיבי מחייב שרף זיקה עם רצון לשמור כולה במזג אוויר פתרון פיזיולוגי. איזון זה משמש כדי לשמר אינטראקציה יציבה (ים) של החלבון מתויג עם אינטראקצית גורם (ים) לאפיון שלאחר טיהור. תג TAP משולב genomically הוצב בסוף (C-terminal) של גן מקודדי חלבונים וכלל רצף קידוד calmodulin מחייב פפטיד (CBP) ואחריו חלבון - תוספת של קצת יותר מ -20 kDa אל מתויג חֶלְבּוֹן. CBP הוא קצר, 26 חומצות אמינו, ומוכר על ידי ~ 17 kDa חלבון calmodulin (CAM) בנוכחות של סידן עם K D בסדר גודל של 10 -9 M 4. תג החלבון גדול, מורכב משתי חזרות של 58 שאריות עם מקשר קצר בין החזרות. כל חוזרות חומצה 58 אמינו הוא מוכר על ידי אימונוגלובולין G (IgG) עם -8 M 5 10 K D ~. בין שני אלה תגים התאגד אתר הכרת פרוטאז TEV, גידול endopeptidase מנגיף הטבק Etch 6,7. כמו באיור 1, בשלב זיקה הראשון של שיטת TAP מתויג חלבון מאוגד שרף IgG דרך א חלבון החלבון מתויג הוא eluted ידי על מחשוף טור על תוספת של פרוטאז TEV, אתר ספציפי ביקוע בין את שני התגים. זהו צעד הכרחי כאינטראקציה של IgG ו- A החלבון הוא חזק מאוד והוא יכול להיות רק מוטרד כראוי בתנאי פתרון denaturing. בהעדר Protein תג, החלבון הוא כבול שרף CAM בנוכחות סידן eluted משרף זה עם תוספת של EGTA chelator מתכת יון (חומצה tetraacetic אתילן גליקול) (איור 1). זמן קצר לאחר כניסתה של שיטת TAP, היא שימשה במחקר בקנה מידה גדול כדי ליצור &quot;מפה&quot; של יחסי הגומלין המורכבים ב ס cerevisiae 8. חשוב לציין, כתוצאה ממאמץ זה המסגרת כולו שמר-TAP Tagged להרחיב קריאה (ORF) ספרייה וכן TAP פרט מתויג ORFs 9 זמינים ממקור מסחרי. לכן, אפשר להשיג כל זן שמרים עם חלבון מתויג לכל מורכבות שמרים. השיטה TAP גם דירבן שינויים או וריאציות של תג TAP, כולל: השימוש בו לטיהור מתחמי מ איקריוטיים אחרים, כמו גם תאים חיידקיים 10,11; העיצוב של &quot;תג לפצל&quot;, שבה החלבון ו CBP מונחים על חלבונים שונים 12; ואת taGS השתנה, כדי להתאים את הצורך של החוקר, כגון רגישות של המתחם כדי Ca 2 + או EGTA 13. ההתקדמות שחלה באחרונה בשני המכשור ומתודולוגיה הובילו להתקדמות משמעותית ביישום של מיקרוסקופיה אלקטרונית (EM) לקביעת מבנה, שהובילו גבוהה, ליד תמונות ברזולוציה אטומית של מתחמי macromolecular 14. החלטת ההשגה של קומפלקס א&quot;מ, לעומת זאת, נותרת מותנית באיכות של המתחם נחקר. מחקר זה נצל את גישת תג TAP לטהר מ ס cerevisiae מספר U1 snRNP, א-למקטע 18 (~ 0.8 מד&quot;א) מורכב ribonucleoprotein נמוך עותק מספר כי הוא חלק 15,16 הספלייסוזום. מספר הצעדים ננקט כדי לטהר מורכב זה כזה שזה הומוגנית של ריכוז נאות. בעיות פוטנציאליות נתקלו בשלבים שונים של הטיהור מתוארות ואסטרטגיות נלקחו להתגבר challenges הדגיש. לפי הערכה ואופטימיזציה צעדים בזהירות הטיהור, U1 snRNP מטוהר הוא באיכות ובבית בכמות שנכנסה כתם שלילי אלקטרוני cryo מיקרוסקופיה (cryo-EM) מחקרים. פרוטוקול שיטת TAP אופטימיזציה עבור טיהור של מתחמי מקורית עבור מחקרים מבניים מתואר במסמך זה.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>S. cerevisiae TAP tagged strain</td>
			<td>Open Biosystems</td>
			<td>YSC1177</td>
			<td>This is the primary yeast strain used to develop the TAP protocol. Its background is S288C: ATCC 201388: MATa, his3&#916;1, leu2&#916;0, met15&#916;0, ura3&#916;0, SNU71::TAP::HIS3MX6</td>
		</tr>
		<tr>
			<td>Coffee grinder</td>
			<td>Mr. Coffee</td>
			<td>IDS77</td>
			<td>Used for cell lysis</td>
		</tr>
		<tr>
			<td>Hemocytometer, Bright Line</td>
			<td>Hausser Scientific</td>
			<td>3120</td>
			<td>Used to assess cell lysis</td>
		</tr>
		<tr>
			<td>JA 9.100 centrifuge rotor&#160;</td>
			<td>Beckman Coulter, Inc.</td>
			<td></td>
			<td>Used to harvest the yeast cells</td>
		</tr>
		<tr>
			<td>JA 20 fixed-angle centrifuge rotor</td>
			<td>Beckman Coulter, Inc.</td>
			<td></td>
			<td>Used to clear the cell extract of non-soluble cellular material</td>
		</tr>
		<tr>
			<td>Ti 60 fixed-angle centrifuge rotor</td>
			<td>Beckman Coulter, Inc.</td>
			<td></td>
			<td>Used to further clear the soluble cell extract</td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Eppendorf</td>
			<td>R5355</td>
			<td>Temperature controlled shaker</td>
		</tr>
		<tr>
			<td>Novex gel system</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IgG resin</td>
			<td>GE Healthcare</td>
			<td>17-0969-01</td>
			<td>Sepharose 6 fast flow</td>
		</tr>
		<tr>
			<td>Calmodulin resin</td>
			<td>Agilent Technologies, Inc.</td>
			<td>214303</td>
			<td>Affinity resin</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail, mini tablets</td>
			<td>Sigma Aldrich</td>
			<td>589297</td>
			<td>Mini cOmplete ultra EDTA-free tablets</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail, large tablets</td>
			<td>Sigma Aldrich</td>
			<td>5892953</td>
			<td>cOmplete ultra EDTA-free tablets</td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride (PMSF)</td>
			<td></td>
			<td></td>
			<td>Dissolved in isopropanol</td>
		</tr>
		<tr>
			<td>2 mL Bio-spin column</td>
			<td>Bio-Rad Laboratories, Inc.</td>
			<td>7326008</td>
			<td>Used to pack and wash the Calmodulin resin</td>
		</tr>
		<tr>
			<td>10 mL poly-prep column</td>
			<td>Bio-Rad Laboratories, Inc.</td>
			<td>7311550</td>
			<td>Used to pack and wash the IgG resin</td>
		</tr>
		<tr>
			<td>Precast native PAGE Bis-Tris gels</td>
			<td>Life Technologies</td>
			<td>BN1002</td>
			<td>Novex NativePAGE Bis-Tris 4-16% precast polyacrylamide gels</td>
		</tr>
		<tr>
			<td>NativeMark protein standard</td>
			<td>Thermo Fisher Scientific</td>
			<td>LC0725</td>
			<td>Unstained protein standard used for native PAGE. Load 7.5 &#956;L for a silver stained gel and 5 &#956;L for a SYPRO Ruby stained gel</td>
		</tr>
		<tr>
			<td>Precast SDS PAGE Bis-Tris gels</td>
			<td>Life Technologies</td>
			<td>NP0321</td>
			<td>Novex Nu-PAGE Bis-Tris 4-12% precast polyacrylamide gels&#160;</td>
		</tr>
		<tr>
			<td>PageRuler protein standard</td>
			<td>Thermo Fisher Scientific</td>
			<td>26614</td>
			<td>Unstained protein standard used for Western blotting</td>
		</tr>
		<tr>
			<td>SDS running buffer</td>
			<td>Life Technologies</td>
			<td>NP0001</td>
			<td>1x NuPAGE MOPS SDS Buffer</td>
		</tr>
		<tr>
			<td>TAP antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>CAB1001</td>
			<td>Primary antibody against CBP tag</td>
		</tr>
		<tr>
			<td>Secondary antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>31341</td>
			<td>Goat anti-rabbit alkaline phosphatase conjugated</td>
		</tr>
		<tr>
			<td>BCIP/NBT</td>
			<td>Thermo Fisher Scientific</td>
			<td>34042</td>
			<td>5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium&#160;</td>
		</tr>
		<tr>
			<td>Dialaysis units</td>
			<td>Thermo Fisher Scientific</td>
			<td>88401</td>
			<td>Slide-A-Lyzer mini dialysis units</td>
		</tr>
		<tr>
			<td>Centrifugal filter units, 100kDa MWCO</td>
			<td>EMD Millipore</td>
			<td>UFC5100008</td>
			<td>Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-100 membrane</td>
		</tr>
		<tr>
			<td>Detergent absorbing beads</td>
			<td>Bio-Rad Laboratories, Inc.</td>
			<td>1523920</td>
			<td>Bio-bead SM-2 absorbants</td>
		</tr>
		<tr>
			<td>SYBR Green II</td>
			<td>Thermo Fisher Scientific</td>
			<td>S-7564</td>
			<td>Flourescent dye for nucleic acid staining, when detecting with SYPRO Ruby present, use excitation wavelength of 488 nm and emission wavelength of 532 nm</td>
		</tr>
		<tr>
			<td>SYPRO Ruby</td>
			<td>Molecular Probes</td>
			<td>S-12000</td>
			<td>Flourescent dye for protein staining, when detecting with SYBR Green II present, use excitation wavelength of 457 nm and emission wavelength of 670 nm</td>
		</tr>
		<tr>
			<td>Copper grids</td>
			<td>Electron Microscopy Sciences</td>
			<td>G400-CP</td>
			<td></td>
		</tr>
	</tbody>
</table>

purification of native complexes for structural study using a tandem affinity tag method

גישות טיהור זיקה הצליחו לבודד מתחמי יליד לאפיון proteomic. ההטרוגניות מבנית ומידה של ההטרוגניות הלחנה של קומפלקס בדרך כלל לא לעכב את ההתקדמות בביצוע מחקרים כאלה. לעומת זאת, קומפלקס המיועד אפיון מבניים צריך להיטהר בתוך מדינה שהיא גם בעלי הרכב הומוגני מבחינה מבנית כמו גם בריכוז גבוה מהנדרש עבור חקר החלבונים. לאחרונה, חלה התקדמות משמעותית ביישום של מיקרוסקופיה אלקטרונית לקביעת מבנה של קומפלקסי macromolecular גדולים. זה הגביר עניין גישות לטהר מתחמי יליד באיכות מספקת וכמות לקביעה מבנית על ידי מיקרוסקופי אלקטרונים. טיהור זיקה טנדם (TAP) השיטה בצורה מיטבית כדי לחלץ ולטהר 18-למקטע, ~ 0.8 הרכבה ribonucleoprotein מד&quot;א שמרים ניצני (שמר האפייה) </em&gt; מתאים כתם שלילי מיקרוסקופיה קריו אלקטרונים. במסמך זה מפורט את השינויים שבוצעו בשיטת TAP, רציונל ביצוע שינויים אלה, ואת הגישות שננקטו כדי assay קומפלקס בעלי רכב המבני הומוגנית.

שיטת TAP שונה שמשה לטהר מ ס cerevisiae מספר U1 snRNP, תסביך ribonucleoprotein 18-למקטע. טיהור TAP ראשונית של המתחם בעקבות 2,3 הפרוטוקול שפורסם ניב קומפלקס שהופיע הטרוגנית, נודדות כמו שלוש להקות על כסף מוכתמות ג&#39;ל polyacrylamide ילידים (איור 2 א). סבבים רבים של ...

Watch this Scientific Journal Video about Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method at JoVE.com

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

טיהור של מכלולי Native לחקר מבני בשיטת תג טנדם זיקה

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree ...

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9.5K Views.  Brandeis University. The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies. 

Video: Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

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 ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

בידוד טיהור תסיסנית נוירונים היקפיים על ידי מיון ביד מגנטי

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

ניתוח מערכות חלבון גדול על ידי ספקטרומטריית מסה מבנית

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

ג&#39;ל כחול Native polyacrylamide אלקטרופורזה (BN-PAGE) עבור ניתוח של מתחמי multiprotein מ lysates נייד

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

חלבון טיהור אורתוגונלית בהנחיית תג זיקה קטנים Bispecific

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

זיהוי שותפים חלבון אינטראקציה באמצעות טיהור טנדם זיקה

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

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 ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

טיהור תפוקה גבוהה של חלבונים רקומביננטי זיקה מתויגת-

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness1. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)2,3 and a C-terminal 10xHis tag4, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression5. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme6. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.

In the present protocol, we demonstrate a highly efficient and cost-effective small-scale protein purification method, which allows purification of recombinant proteins by uniquely combining a cleavable GST-tag and a small His-tag.

GST-טיהור: שני שלבי זיקה טיהור פרוטוקול מניב חלבונים מטוהרים באורך מלא

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

שיטה ללא טיהור חלבונים של קביעת זיקה מחייבת ידי thermophoresis Microscale

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...