פרויקט זה נתמך על ידי מענקים CJJ מן הקרן הלאומית למדע והחברה למדעים Biomolecular.

1. יום 1: MexB מ Pseudomonas aeruginosa מקודד על ידי pFB101. הגן MexB הוגדל מ - פ aeruginosa DNA הגנומי ונוסף על NdeI ואתרי XhoI הגבלה של + pET30b וקטור. לבנות מכיל תג בטרמינל C-hexahistidine. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> פלסמיד משמש כדי להפוך את ה coli זן C43 (DE3) 12, ואת transformants הם מצופה על אגר LB המכיל 30 UG / mL kanamycin. </li></ol> 2. יום 2: תרבויות לינה: <ol start="2" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בערב, 4 X 3 LB המכיל 30 מ&quot;ל תרבויות UG / mL kanamycin הם מחוסן מן המושבות transformant טריים. לחלופין, תרבויות יכול להיות מחוסן מפני סלסול קפוא. תרבויות אלו קטנים הם גדלו על מכבש על 37 מעלות צלזיוס למשך הלילה. </li></ol> 3. יום 3: גידול 6 ליטר תרבויות: <ol start="3" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בבוקר, השתמש תרבויות לילה לחסן LB 150 מ&quot;ל המכיל 30 UG / mL kanamycin. לגדול התרבות ב 37 ° C על שייקר. </li><li style=";text-align:right;direction:rtl"> בשעות אחר הצהריים, להשתמש תרבות קטן לחסן 6 x 1 ליטר 2XYT מדיה המכילה 30 UG / kanamycin מ&quot;ל צלוחיות Fernbach. (השתמש 25 מ&quot;ל לכל תרבות לדילול 01:40). לגדול התרבויות ב 37 מעלות צלזיוס עד שהם מגיעים OD של 600 0.4-0.6, כ -1.5 שעות </li><li style=";text-align:right;direction:rtl"> כאשר התרבויות להגיע צפיפות נכונה, להשרות ביטוי החלבון על ידי הוספת 0.5 מ&quot;ל 1M IPTG. מכניסים את כל צלוחיות בשנות ה שייקר ולהמשיך לגדל אותם ב 30 מעלות צלזיוס למשך הלילה. </li></ol> 4. יום 4: קצירת התאים וטיהור חלבון: <ol start="6" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הוסף מעכבי פרוטאז, DNAse ו ליזוזים לפתרונות חיץ כדלקמן: עד 50 מ&quot;ל של חיץ resuspension התא, להוסיף 10 מ&quot;ג DNaseI (0.1 מ&quot;ג / מ&quot;ל ​​בריכוז הסופי), 1 מעכב השלם EDTA ללא פרוטאז הלוח, וקורט ליזוזים . עד 60 מ&quot;ל של חיץ resuspension קרום, להוסיף 1 מעכבי פרוטאז הלוח. כדי 50 מ&quot;ל נוסף של המאגר resuspension קרום, להוסיף 1 מעכבי פרוטאז הלוח. שמור את כל שלושה פתרונות על הקרח. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה התרבויות 30 דקות 5,000 סל&quot;ד בצנטריפוגה בקנה מידה גדול כדי לקצור את התאים. </li><li style=";text-align:right;direction:rtl"> Resuspend התאים חיץ תא resuspension 100 מ&quot;ל (50 NAP מ&quot;מ, pH 7.0, 300 mM NaCl, 2 מ&quot;מ MgCl 2, 1 מעכב השלם EDTA ללא לוח פרוטאז, 0.1 מ&quot;ג / מ&quot;ל DNAse אני, קמצוץ של ליזוזים) </li><li style=";text-align:right;direction:rtl"> לעבור את פתרון התא פעמיים באמצעות תא הלחץ הצרפתי 12,000 psi (762 מד הלחץ). אסוף את lysate תא בקבוק קר כל הזמן על הקרח. </li><li style=";text-align:right;direction:rtl"> מעבירים את lysate התא SS34 צינורות צנטריפוגה ו צנטריפוגות כדי להסיר שאריות תאים למשך 30 דקות בסל&quot;ד 10,000 ב 4 מעלות צלזיוס הרוטור SS-34. </li><li style=";text-align:right;direction:rtl"> הסר בזהירות את supernatant לתוך צינורות Ti647.5 ultracentrifuge. צנטריפוגה 50 דקות בסל&quot;ד 40,000 ב 4C. בטל supernatant. </li><li style=";text-align:right;direction:rtl"> Resuspend גלולה, אשר מכיל את קרום התא, על כ. 25 מ&quot;ל של חיץ resuspension קרום (50 NAP מ&quot;מ, pH 7.0, 300 mM NaCl, גליצרול 5%, 1 מעכב השלם EDTA ללא לוח פרוטאז). </li><li style=";text-align:right;direction:rtl"> מעבירים את ההשעיה קרום לצינור צנטריפוגה צנטריפוגה נקי ב 40,000 סל&quot;ד ב הרוטור Ti647.5 עבור 50 דקות ב 4 ° C. </li><li style=";text-align:right;direction:rtl"> בטל supernatant ו resuspend הממברנה גלולה לרחוץ 25 חוצץ קרום מ&quot;ל resuspension (50 NAP מ&quot;מ, pH 7.0, 300 mM NaCl, גליצרול 5%, 1 מעכב השלם EDTA ללא לוח פרוטאז). </li></ol> 5. TM Solublization חלבון: <ol start="15" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי הממברנות resuspended (כ 25 מ&quot;ל), מוסיפים 6 DDM מ&quot;ל 10% (ריכוז חומר הניקוי הסופי = DDM 2%) רוק את התערובת על 4 מעלות צלזיוס למשך 2 שעות. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה את התערובת בסל&quot;ד 40,000 עבור 40 דקות על 4 מעלות צלזיוס הרוטור Ti647.5 כדי להפריד אבקת חלבון מסיס קומפלקסים של חלבונים מסיסים. שמור את supernatant, המכיל אבקת חלבון מתחמי MexB. </li></ol> 6. IMac: <ol start="17" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מערבבים את supernatant המתקבל ספין במהירות גבוהה עם זיקה חרוזי מתכת טופר equilibrated במאגר resuspension. דגירה של 1 שעות על רולר בבית 4 ° C. </li><li style=";text-align:right;direction:rtl"> יוצקים את התערובת לתוך גוף זרימת הכבידה טור וזורקים את הזרימה דרך. </li><li style=";text-align:right;direction:rtl"> לשטוף את הטור עם 20 מ&quot;ל (10 כרכים עמודה) של הצפת מחייב לשטוף iMac (50 NAP מ&quot;מ, ה-pH 7, 300 mM NaCl, גליצרול 5%, DDM 0.2%) </li><li style=";text-align:right;direction:rtl"> Elute את החלבון עם חיץ elution iMac (50 NAP מ&quot;מ, pH 7.0, 300 mM NaCl, גליצרול 5%, 250 מ&quot;מ imidazole, DDM 0.2%). </li><li style=";text-align:right;direction:rtl"> קח 15 דגימות μL של שברים elution, לערבב עם כל 15 חיץ μL SDS 2X מדגם לניתוח על ידי ג&#39;ל אלקטרופורזה polyacrylamide. ספין 30sec ב microcentrifuge. ניתוח דגימות על polyacrylamide של 10% SDS ג&#39;ל לאמוד את הסכום ואת טוהר MexB בשבריר אחד. </li><li style=";text-align:right;direction:rtl"> בריכת שברים המכיל את אבקת חלבון מתחמי MexB ולרכז אותם concentrator ספין ב 4 ° C. היזהר כי החלבון אינו המשקע החוצה זה sטפ. </li></ol> 7. סינון ג&#39;ל עמודה: <ol start="23" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> טרום לאזן 12 Superose HL 30/10 טור עם חיץ 24 מ&quot;ל ריצה (50 NAP מ&quot;מ, pH 7.0, 300 mM NaCl, גליצרול 5%, 0.2% בטא octylglucoside), ולחכות הבסיס שטוח. </li><li style=";text-align:right;direction:rtl"> שוטפים את טעינת מערכת אקטע לולאה עם חיץ פועל. </li><li style=";text-align:right;direction:rtl"> סנן הפתרון חלבון באמצעות מסנן מזרק לפני החלת אותו הטור. </li><li style=";text-align:right;direction:rtl"> טען עד 240 μL של הפתרון חלבון על הטור, עם ריכוז חלבון של עד 5 מג / מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> הפעלה 1.5 כרכים עמודה (36 מ&quot;ל) של חיץ, לאסוף את השברים 0.25 מ&quot;ל. אבקת חלבון מתחמי MexB צריך elute כמו לשיא בסביבות 10-15 מ&quot;ל של נפח elution. </li><li style=";text-align:right;direction:rtl"> קחו 5 דגימות μL של שברים שיא. מערבבים את כל מדגם 01:01 עם חיץ מדגם SDS 2X. ניתוח דוגמאות על ג&#39;ל polyacrylamide 10% כדי לאמוד את הסכום ואת טוהר MexB בשבריר כל </li><li style=";text-align:right;direction:rtl"> בריכת שברים המכיל MexB טהור. </li></ol> 8. נציג תוצאות: איור 1 כוללת ג&#39;ל polyacrylamide עם שברים טור נקווה מהעמודה iMac ו שברים בודדים מעמודה הסינון ג&#39;ל. אחרי הטור סינון ג&#39;ל חלבון מופיע טהור על ידי ג&#39;ל polyacrylamide Coomassie מוכתם. איור 2 כולל עקבות מהעמודה ג&#39;ל סינון מראה את השיא העיקרי של משחררי אבקת חלבון מורכב מהעמודה. התשואה הממוצעת של חלבון MexB הוא כ 2 מ&quot;ג 6 ליטרים של תרבות 2XYT. <img src="/files/ftp_upload/2134/2134fig1.jpg" alt="איור 1" /> באיור 1. SDS-PAGE ג&#39;ל של טיהור PDCs MexB. Lane 1, משקל סמנים מולקולריים. 2, משולב שברים iMac. 3-7, טור ג&#39;ל סינון שברים. <img src="/files/ftp_upload/2134/2134fig2.jpg" alt="איור 2" /> איור 2. דוגמה סינון תוצאות ג&#39;ל ניקוי MexB קומפלקסים חלבונים (PDC).

<ol>
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Bacteriol. 178, 306-308 (1996).</li><li>Srikumar, R., Kon, T., Gotoh, N., Poole, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Pseudomonas+aeruginosa+multidrug+efflux+pumps+MexA-MexB-OprM+and+MexC-MexD-OprJ+in+a+multidrug-sensitive+Escherichia+coli+strain.">Expression of Pseudomonas aeruginosa multidrug efflux pumps MexA-MexB-OprM and MexC-MexD-OprJ in a multidrug-sensitive Escherichia coli strain.</a> Antimicrob. Agents Chemother. 42, 65-71 (1998).</li><li>Tikhonova, E. B., Zgurskaya, H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AcrA,+AcrB,+and+TolC+of+Escherichia+coli+Form+a+Stable+Intermembrane+Multidrug+Efflux.">AcrA, AcrB, and TolC of Escherichia coli Form a Stable Intermembrane Multidrug Efflux.</a> Complex. J. Biol. 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בנוסף multidrug, התנגדות פעילויות רבות הסלולר חיוניים, כולל תחבורה ותקשורת יון תאים תאים, תחבורה, שלפוחית, תחזוקה של המבנה התאי, והוא מארח הפתוגן אינטראקציות, לערב חלבונים אשר מוטבעות בתוך קרום התא. חלבונים הטרנסממברני לפצות מעל 30% של חלבונים ידועים הם מטרות עבור רוב התרופות בשימוש כיום. קיפול לא נכון או פעילות של חלבונים הטרנסממברני להוביל מחלות גנטיות חשובות, כולל סיסטיק פיברוזיס סוכרת ו. למרות החשיבות העצומה של חלבונים הטרנסממברני, יש הרבה פחות ידוע על המבנים שלהם ואת המנגנונים המולקולריים מאשר חלבונים מסיסים. נוכחות של רצפי הידרופובי יכול להקשות לבטא לבודד כמויות גדולות של חלבונים אלו והופך אותם עקשן שיטות ביוכימיות מבניים רבים. פרוטוקול זה הוכיח את הבעת פניו חומר ניקוי solubilization וטיהור של חלבון קרום MDR כמו קומפלקס חלבון מסיס חומר ניקוי. שיטות אלה ניתן להשתמש עם כמה שינויים רבים חלבונים הטרנסממברני הביע recombinantly. כתוצאה מכך מטוהרים ניקוי קומפלקסים חלבונים מסיסים הם יכולים לשמש בניסויים התגבשות להגדרה רנטגן מבנה קריסטלוגרפיים ו לאפיון biophysical או ביוכימיים אחרים, כולל הכינון מחדש לתוך ליפוזומים או מחקרים crosslinking. במהלך הליכי טיהור, חשוב להיות זהירים כי חומר ניקוי קומפלקסים חלבונים לא להאיץ החוצה במהלך השלבים ריכוז ספין. שיטות שונות עשוי לשמש גם כדי לעזור לרכז את PDC לפני או אחרי צעד את הג&#39;ל סינון, כגון חזרה על צעד iMac עם טור קטן מאוד ונפח elution קטן.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>SDS sample buffer</td>
<td>&nbsp;</td>
<td>Biorad</td>
<td>161-0737</td>
<td>&nbsp;</td>
<tr>
<td>C43(DE3) E. coli strain</td>
<td>&nbsp;</td>
<td>Lucigen</td>
<td>60345-1</td>
<td>&nbsp;</td>
<tr>
<td>kanamycin sulfate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>K4378</td>
<td>&nbsp;</td>
<tr>
<td>2XYT media</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP2466-2</td>
<td>&nbsp;</td>
<tr>
<td>LB media</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>AC61189-5000</td>
<td>&nbsp;</td>
<tr>
<td>IPTG</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>I6758</td>
<td>&nbsp;</td>
<tr>
<td>DNaseI</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP3226-1</td>
<td>&nbsp;</td>
<tr>
<td>Lysozyme</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>L7651</td>
<td>&nbsp;</td>
<tr>
<td>Complete EDTA-free protease inhibitor tablets</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11 873 580 001</td>
<td>&nbsp;</td>
<tr>
<td>NaP monobasic</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S6566</td>
<td>&nbsp;</td>
<tr>
<td>NaP dibasic</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S5136</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S6191</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M1028</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP229-1</td>
<td>&nbsp;</td>
<tr>
<td>n-dodecyl-&beta;-D-maltopyranoside</td>
<td>&nbsp;</td>
<td>Anatrace</td>
<td>D310</td>
<td>&nbsp;</td>
<tr>
<td>15ml tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430052</td>
<td>&nbsp;</td>
<tr>
<td>See-Saw Rocker</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SSL 4</td>
<td>&nbsp;</td>
<tr>
<td>Talon metal affinity resin</td>
<td>&nbsp;</td>
<td>Clontech</td>
<td>635503</td>
<td>&nbsp;</td>
<tr>
<td>imidazole</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>I5513</td>
<td>&nbsp;</td>
<tr>
<td>10% polyacrylamide SDS PAGE gels</td>
<td>&nbsp;</td>
<td>BioRad</td>
<td>161-1454</td>
<td>&nbsp;</td>
<tr>
<td>Tris/glycine/SDS PAGE running buffer</td>
<td>&nbsp;</td>
<td>BioRad</td>
<td>161-0732</td>
<td>&nbsp;</td>
<tr>
<td>Kaleidascope prestained molecular weight markers</td>
<td>&nbsp;</td>
<td>BioRad</td>
<td>161-0324</td>
<td>&nbsp;</td>
<tr>
<td>Superose 12 30/10 column</td>
<td>&nbsp;</td>
<td>GEHealthcareSuperose</td>
<td>12 10/300 GL</td>
<td>&nbsp;</td>
<tr>
<td>Amicon centrifugal concentrator</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>UFC801024</td>
<td>&nbsp;</td>
<tr>
<td>Syringe filter</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SLFG R04 NL</td>
<td>&nbsp;</td>
<tr>
<td>Fernbach flasks</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>09-552-39</td>
<td>&nbsp;</td>
<tr>
<td>Shaker to hold Fernbach flasks</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Akta system</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>J6 Large scale centrifuge with JLA-8.1000 rotor</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1 l centrifuge bottles</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>969329</td>
<td>&nbsp;</td>
<tr>
<td>RC-5 centrifuge</td>
<td>&nbsp;</td>
<td>ThermoScientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>SS34 fixed-angle rotor and tubes</td>
<td>&nbsp;</td>
<td>ThermoScientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sorvall floor model Ultracentrifuge</td>
<td>&nbsp;</td>
<td>ThermoScientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>T647.5 rotor and tubes with caps</td>
<td>&nbsp;</td>
<td>ThermoScientific</td>
<td>08322</td>
<td>&nbsp;</td>
<tr>
<td>French Pressure Cell</td>
<td>&nbsp;</td>
<td>ThermoScientific</td>
<td>FA-032</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

expression, detergent solubilization, and purification of a membrane transporter, the mexb multidrug resistance protein

התנגדות Multidrug (MDR), היכולת של תא סרטני או פתוגן להיות עמידים בפני מגוון רחב של תרופות נוגדות סרטן מבנית או תפקודית ואינו קשור אנטיביוטיקה, היא בעיה רצינית הנוכחי בריאות הציבור. זו התנגדות multidrug נובע במידה רבה אנרגיה תלויי משאבות בזרימת סמים. המשאבות לגרש תרופות נוגדות סרטן או אנטיביוטיקה לתוך המדיום חיצוניים, הורדת ריכוז תאיים שלהם מתחת לסף רעילים. אנחנו לומדים התנגדות multidrug ב Pseudomonas aeruginosa, פתוגן חיידקי אופורטוניסטית שגורם לזיהומים בחולים עם סוגים רבים של פציעה או מחלה, למשל, כוויות או סיסטיק פיברוזיס, וגם חיסונית נפגעת דיאליזה סרטן, חולים להשתלה. בזרימת הגדולות MDR משאבות פ aeruginosa הם קומפלקסים משולשת המורכבת antiporter הפנימית הממברנה פרוטונים סמים (RND), ערוץ הממברנה החיצונית (OMF), לבין חלבון מקשר periplasmic (MFP) 1-8. החלבונים RND ו OMF הם חלבונים הטרנסממברני. חלבונים הטרנסממברני לפצות יותר מאשר 30% של כל החלבונים הם 65% מטרות תרופה הנוכחי. תחומים הטרנסממברני הידרופובי לייצר חלבונים מסיסים חיץ מימית. לפני חלבון הטרנסממברני יכול להיות מטוהרים, יש צורך למצוא את התנאים חוצץ המכיל חומר ניקוי עדין המאפשרים את החלבון להיות solubilized כמתחם אבקת חלבון (PDC) 11/09. בדוגמה זו, אנו משתמשים חלבון RND, פ MexB aeruginosa טרנספורטר הטרנסממברני, כדי להדגים איך לבטא צורה רקומביננטי של חלבון הטרנסממברני, solubilize אותו באמצעות דטרגנטים, ולאחר מכן לטהר את אבקת קומפלקסים חלבונים. שיטה זו בכלל ניתן ליישם את הביטוי, טיהור, ו solubilization של חלבונים רבים אחרים קרום הביע recombinantly. ניקוי קומפלקסים חלבונים יכול מאוחר יותר לשמש אפיון ביוכימי או biophysical לרבות קביעת רנטגן קריסטל מבנה או מחקרים crosslinking.

Watch this Scientific Journal Video about Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein at JoVE.com

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

בפרוטוקול הזה אנחנו מדגימים את הביטוי, solubilization, וטיהור של חלבון קרום הביע recombinantly, MexB, כפי מורכב חלבון מסיס חומר ניקוי. MexB היא התנגדות הממברנה multidrug טרנספורטר מן החיידק Pseudomonas aeruginosa אופורטוניסטית הפתוגן.

הביטוי, solubilization דטרגנט, ועל טיהור Transporter ממברנה, חלבון ההתנגדות MexB Multidrug

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated ...

expression-detergent-solubilization-purification-membrane-transporter

Nanyang Technological University

Research

JoVE Journal

Biology

21.2K Views.  University of Illinois Chicago - UIC. בפרוטוקול הזה אנחנו מדגימים את הביטוי, solubilization, וטיהור של חלבון קרום הביע recombinantly, MexB, כפי מורכב חלבון מסיס חומר ניקוי. MexB היא התנגדות הממברנה multidrug טרנספורטר מן החיידק Pseudomonas aeruginosa אופורטוניסטית הפתוגן.

Video: Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis

Patients with active pulmonary tuberculosis (TB) infect 10-15 other persons per year, making diagnosing active TB essential to both curing the patient and preventing new infections.  Furthermore, the emergence of multidrug resistant tuberculosis (MDRTB) means that detection of drug resistance is necessary for stopping the spread of drug-resistant strains.  The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of TB and MDRTB.  The MODS assay is based on three principles: 1) mycobacterium tuberculosis (MTB) grows faster in liquid media than on solid media 2) microscopic MTB growth can be detected earlier in liquid media than waiting for the macroscopic appearance of colonies on solid media, and that growth is characteristic of MTB, allowing it to be distinguished from atypical mycobacteria or fungal or bacterial contamination 3) the drugs isoniazid and rifampicin can be incorporated into the MODS assay to allow for simultaneous direct detection of MDRTB, obviating the need for subculture to perform an indirect drug susceptibility test. Competing current diagnostics are hampered by low sensitivity with sputum smear, long delays until diagnosis with solid media culture, prohibitively high cost with existing liquid media culture methods, and the need to do subculture for indirect drug susceptibility testing to detect MDRTB.  In contrast, the non-proprietary MODS method has a high sensitivity for TB and MDRTB, is a relatively rapid culture method, provides simultaneous drug susceptibility testing for MDRTB, and is accessible to resource-limited settings at just under $3 for testing for TB and MDRTB.

The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of tuberculosis (TB) and multidrug-resistant tuberculosis (MDRTB). This video describes the MODS liquid media culture method.

השיטה MODS לאבחון שחפת שחפת עמידים multidrug

Patients with active pulmonary tuberculosis (TB) infect 10-15 other persons per year, making diagnosing active TB essential to both curing the patient and ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

הכתמה של חלבונים עם ג&#39;ל Coomassie G-250 ללא חומצה אצטית ממס אורגני

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

שימוש ג&#39;ין Pulser מערכת MXcell electroporation כדי Transfect תאים ראשוניים עם יעילות גבוהה

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

טיהור של מ magneticum מסננים השגריר-1 Magnetosome חלבון Associated MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

שימוש מונה תא אוטומטי כדי לפשט את הביטוי מחקרים ג&#39;ין: siRNA מציאה הביטוי IL 4-Gene תלויים תאים Namalwa

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

טיהור Hsp104, Disaggregase חלבון

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

ביטוי וטיהור של חלבון פיברוזיס פיברוזיס הטרנסממברני הרגולטור מוליכות ב Saccharomyces cerevisiae

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

הפקת ניצני שמרים וטיהור חלבון לחקר תפקוד, האינטראקציות, ולאחר translational השינויים

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

הקרנת siRNA לזיהוי יוביקוויטין ורגולטורים מערכת דמויות יוביקוויטין של מסלולים ביולוגיים בתאי יונקים בתרבית

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

הקרנה ירוקה פלורסנט מבוסס חלבון ביטוי של החלבונים ממברנה ב<em&gt; Coli Escherichia</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...