עבודה זו מומנה על ידי NEURON-Eranet גלה (BMBF 01EW1003) לCK וJPH, DFG (Ko 1679/3-1; GRK1033) לCKVB נתמך על ידי מענק של Forschungskommission של הפקולטה לרפואה באוניברסיטת דיסלדורף.

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אין ניגודי האינטרסים הכריזו.

במחקר זה אנו מתארים טיהור mRFP/eGFP-DISC1 aggresomes מקו transfected נוירובלסטומה תא, ההכנה ותיוג של DISC1 מיני חלבון רקומביננטי והיישום שלהם בניסויי תא פלישה של תאי נמען במבחנת in vivo. טיהור mRFP/eGFP-DISC1 aggresomes ילידים פותחה מתוך פרוטוקולים לבודד מבנים לוי גוף דמויים ואגרגטים גדולים 13, 14, אבל שונה כדי להימנע מהשימוש בחומרי ניקוי וכדי למזער את הסיכון של תא פלישה חיובית כוזב שעלולה להתרחש עקב חדירת חומר ניקוי קרום הקל. שיפור עתיד אפשרי אחד יכול להיות בהמשך להגדיל את הטוהר של aggresomes ידי sonication ללחלוטין קרומים שנותרו נפרדים וcytoskeleton מaggresomes. על ידי טוהר כולל גובר, מספר אירועי פלישה כלל שנרשם לaggresomes ma גדולy להיות מוגבר 5. אם בהגדרה המצומצמת של סוכרוז הציע 3-השלב די לaggresomes של מיני חלבון אחרים יש להיבדק, במובן זה הפרוטוקול המתואר כאן צריך להיחשב כנקודה מוצאת לקידום פרט. את aggresomes המטוהר הוכיח להיות די חזק בידות שלנו, שלבי כביסה נוספים (ללא חומרי ניקוי) לחסל את העקבות של סוכרוז לא להפחית באופן משמעותי את התשואה הכוללת. בפרסום קודם שתארנו שהרכבת oligomer של DISC1 תלויה בתחומי multimerization נפרדים בתחנה הסופית של C-4 (איור 4). בחרנו קטע זה לביטוי רקומביננטי המסיס בE. coli ושלאחר מכן וטיהור Ni-נ.ת. ע. כדי לפקח multimerization ולהשוות את הפולשנות התא שלה עם חלבון תאר תא פולשנית כמו α-סינוקלאין, אנחנו מתויגים DISC1 (598-785) עם צבע פלואורסצנטי DyLight594, ולעומתהתא הפולשנות עם זה של תווית באופן שווה α-סינוקלאין. תא הפולשנות של DISC1 בר רקומביננטי הייתה גדל באופן דרמטי בהשוואה לזה של ילידי אורך מלא DISC1 aggresomes, כנראה עקב גודל קטן יותר וטוהר גבוה בהרבה. שוב, טוהר נראה לשחק תפקיד מכריע בתא הפולשנות. בדוגמא שלנו את aggresomes פולשניים גייס חלבון מסיס homologue של קו תא היעד שהתבטא recombinantly בצורה מסיסה, כלומר תא מפוזר-. הביטוי של חלבון פלואורסצנטי (למשל GFP או RFP) בשורת תאי הנמען הוא יתרון שכן הוא מאפשר colocalization של aggresomes פולשניים וחלבונים רקומביננטיים בתוך מגבלת תא הנמען באמצעות הדמית Z-מחסנית. aggresomes DISC1 תאים פולשניים ושברי multimeric הביעו ומטוהר מE. חיידק יכול להיות תכונה מגדירה של מחלות קונפורמציה חלבון הנוגעים לDISC1, DISC1opathies 15. צרות קליעה: תשואה נמוכה aggresome לאחר טיהור שיפוע סוכרוז: שיפוע סוכרוז הציג בפרוטוקול זה עובד היטב עם aggresomes eGFP/mRFP-DISC1 (פלורידה). Aggresomes המורכב של חלבונים אחרים עשויה להיות מידות משתנות ולכן ריכוזי סוכרוז צריכים להיות מותאמים על ידי משתמשים אחרים. טיהור בלתי מספקת של חלבון רקומביננטי: כל זיהום חיידקים בתהליך הטיהור של חלבון רקומביננטי גם להיות מתויג בשלבים מאוחרים יותר של הפרוטוקול. כדי להימנע מזיהומים, להפעיל את החלבון על SDS-PAGE ולוודא כי חלבון רקומביננטי הוא לפחות 95% טהור ע&quot;י השאיר עם Coomassie הכחול. כדי להבטיח איכות ואת התשואה הגבוהה ביותר, בפרוטוקול יש להיות מותאם לכל חלבון בודד. נמוך התיוג EFficiency של חלבונים רקומביננטיים: כל זיהומים שמכילים קבוצות SH חופשיות יקטנו תיוג יעיל של החלבון. לכן, דיאליזה נרחבת וטיהור מבוססת Ni-נ.ת. ע הוא חובה.

<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> RPMI 1640 </td><td> Invitrogen </td><td> 11875-093 </td><td> בינוני תלוי בקו התא המארח. שורת תאי הנמען האופטימלית צריכה להיקבע על ידי המשתמש. </td></tr><tr><td> DMEM/F-12 </td><td> Invitrogen </td><td> 11320-033 </td><td> בינוני תלוי בקו תא הנמען. שורת תאי הנמען האופטימלית צריכה להיקבע על ידי המשתמש. </td></tr><tr><td> PBS </td><td> Invitrogen </td><td> 14190-250 </td><td></td></tr><tr><td> Metafectene </td><td> Biontex </td><td> T020-1.0 </td><td> ריאגנטים transfection אחרים יכולים לעבוד גם כן, אבל לא נבדקו בפרוטוקול שלנו. </td></tr><tr><td> Ni-נ.ת. ע agarose </td><td> Qiagen </td><td> 30210 &lt;/ Td&gt; <td> הניסויים נעשו עם Ni_NTA agarose מQiagen, ספקים אחרים צריכים לעבוד גם כן. </td></tr><tr><td> אני DNase </td><td> רוש </td><td> 04716728001 </td><td> אין צורך בRNase החופשי DNase בתהליך של טיהור aggresomes. </td></tr><tr><td> סוכרוז </td><td> סיגמה אולדריץ </td><td> S0389 </td><td></td></tr><tr><td> Opti-ממ </td><td> Invitrogen </td><td> 31985-062 </td><td> מדיום סרום ללא עובד כמו גם </td></tr><tr><td> פניצילין / סטרפטומיצין </td><td> Invitrogen </td><td> 15140122 </td><td> תוספת לSH-SY5Y וNLF הבינוני </td></tr><tr><td> שאינן חיונית, חומצות אמינו (NEAA) </td><td> סיגמה אולדריץ </td><td> M7145 </td><td> תוספת למדיום SH-SY5Y </td></tr><tr><td> Trypan הכחול 0.4% </td><td> סיגמה אולדריץ </td><td> T8154 </td><td> ריאגנט רעיל </td></tr><tr><td> DyLight 594 מאleimide </td><td> תרם פישר סיינטיפיק </td><td> 46608 </td><td> הקטין לצבוע תגובתי ציסטאין ליצור קשרי thioether יציבים. זמין גם בצבעים אחרים. </td></tr><tr><td> להאריך את הזהב עם DAPI </td><td> Invitrogen </td><td> P36935 </td><td> mountant הנוזל antifade זה נתן תוצאות טובות יותר בידות שלנו. </td></tr><tr><td> קוקטייל מעכב פרוטאז </td><td> רוש </td><td> 11873580001 </td><td> ממס טבלה 1 מתוך 500 H 2 O μl לפתרון 100X. </td></tr><tr><td> סינטטי-סינוקלאין </td><td> סיגמא </td><td> S7820 </td><td> לקפל כפי שמתואר בטקסט. </td></tr><tr><td></td><td></td><td></td><td> טבלה של חומרים כימיים ספציפי. </td></tr><tr><td> 24 Precellys </td><td> טכנולוגיות רטין </td><td> 03119.200.RD000 </td><td> אנחנו עדיין לא נבחנו homoge מכאני אחרnizers אחר מזה. שיטת homogenization חופשיה אחרת דטרגנט יכול לעבוד גם כן, אבל לא נבדקה לפרוטוקול זה. </td></tr><tr><td> 5301 מרוכזים (speedvac) </td><td> אפנדורף </td><td> 5301 000.210 </td><td> הכרחי רק אם חלבון צריך להיות מרוכז. </td></tr><tr><td> LSM 510 וAxioVision Apotome2 </td><td> Zeiss </td><td> ראה קטלוג יצרנים </td><td> שני מיקרוסקופי נמל היכולת לבצע הדמית Z-מחסנית. זו היא תנאי הכרחית להערכה מוצקה ורצינית של אירועי פלישה. </td></tr><tr><td> חומר פלסטי תרבית תאים </td><td> Nunc </td><td> 10 מנות סנטימטר # 172958, צלחות 24 # 142475 גם </td><td> השימוש בחומר פלסטי אחר עשוי להשפיע על האינטראקציה של חלבונים רקומביננטיים או aggresomes ואת משטחי הפלסטיק. אנחנו עדיין לא נבחנו חומרים אחרים מאלו שתוארו כאן. </td></tr><tr><td></td><td></td><td></td><td> טבלה של חומרים וציוד ספציפיים. </td></tr><tr><td></td><td></td><td></td><td><table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> מספר </td><td> שם מאגר </td><td> תוכן </td><td> תגובות </td></tr><tr><td> 1. </td><td> מדיום גידול חיידקים </td><td> 16 ג&#39;/ ליטר Bacto Tryptone, תמצית 10 גרם / ליטר Bacto שמרים, 5 גרם / ליטר NaCl, 5 מ&quot;מ ארגינין-HCl, MGSO4 5mm, 100 מיקרוגרם / המ&quot;ל carbencillin, 35 מיקרוגרם / המ&quot;ל chloramphenicol </td><td> לגדול חיידקים לOD 600: 0.6-0.8 לגרום ביטוי עם IPTG 1 מ&quot;מ. </td></tr><tr><td> 2. </td><td> חיץ resuspension קטריאלי </td><td> 50 המ&quot;מ TRIS-HCl pH 8.0, 5 המ&quot;מ EDTA, 2 המ&quot;מ PMSF, 1% TX-100, 250 מיקרוגרם / המ&quot;ל Lysozyme, 20 מ&quot;מ MgCl 2 ו 400 המ&quot;ל DNase U/50 </td><td> Resupend גלולת חיידקים מ1 הליטר oveתרבות rnight במאגר resuspension מ&quot;ל 50. </td></tr><tr><td> 3. </td><td> חיץ מיצוי חלבון </td><td> 50 mM טריס pH 8.0, 5 המ&quot;מ imidazole, 500 המ&quot;מ NaCl, אוריאת ז 8 ו 10 מ&quot;מ ב-ME </td><td></td></tr><tr><td> 4. </td><td> חיץ לשטוף עמודת Ni-נ.ת. ע </td><td> 50 mM טריס pH 8.0, 5 המ&quot;מ imidazole, 500 המ&quot;מ NaCl, אוריאת ז 8 ו 10 מ&quot;מ ב-ME, 12 המ&quot;מ imidazole </td><td> חיץ חילוץ שונה עם 12 המ&quot;מ imidazole הוסיף. </td></tr><tr><td> 5. </td><td> חיץ elution עמודת Ni-נ.ת. ע </td><td> 50 mM טריס, 500 mM NaCl, 300 המ&quot;מ imidazole, ז 8 אוריאה, 1 המ&quot;מ PMSF, 5 מ&quot;מ ו10 mM EDTA ב-ME </td><td> חיץ elution מכיל 10 mM ב-ME, הדיאליזה בהמשך. </td></tr><tr><td> 6. </td><td> PBS </td><td> 8 גרם / הליטר NaCl, 0.2 גר &#39;/ ליטר KCl, 1.44 גר&#39; / ליטר Na 2 HPO 4, 0.24 ג&#39;/ ליטר KH 2 PO 4, להסתגל לpH 7.4 </td><td></td></tr><tr><td>7. </td><td> תיוג חיץ </td><td> PBS המכיל TCEP 5 מ&quot;מ </td><td></td></tr><tr><td colspan="4"> טבלה של מתכונים. </td></tr></tbody></table></td></tr></tbody></table>

generation, purification, and characterization of cell-invasive disc1 protein species

צבירת חלבון נתפסה כסימן היכר כללי של תנאי מוח כרוניים, ניווניות כמו, למשל, במחלות ניווניות מחלת אלצהיימר (Aβ, טאו), מחלת פרקינסון (α-סינוקלאין), מחלת הנטינגטון (polyglutamine, huntingtin), ואחרים. צבירת חלבון היא חשבה להתרחש עקב proteostasis המופרע, כלומר חוסר האיזון בין נובע ופירוק חלבוני misfolded. מן ראוי לציין כי את אותם החלבונים נמצאים מצטברים בצורות ספורדיות של מחלות אלו שמוטציה בגרסות נדירות של צורות משפחתיות. סכיזופרניה היא מחלה כרונית פרוגרסיבית מוח שבמקרים רבים הולכים יחד עם גירעון הקוגניטיבי קבוע ובלתי הפיך. בגישת גן מועמד, אנחנו בדקנו אם 1 שבשנו ב-סכיזופרניה (DISC1), גן משובט במשפחה סקוטית עם הצמדה למחלה כרונית נפשית 1, 2, יכולה להימצא כאגרגטים מסיסים בbrain של מקרים ספוראדיים של סכיזופרניה 3. שימוש CC SMRI, זיהה בכ 20% ממקרים עם CMD אבל לא ביקורת רגילה או חולים עם מחלות ניווניות immunoreactivity DISC1 sarkosyl מסיס לאחר החלוקה ביוכימי. מחקרים מאוחרים יותר במבחנה גילו כי נטיית הצבירה של DISC1 הושפעה פולימורפיזם מחלה הקשורה S704C 4, ושDISC1 aggresomes שנוצר במבחנה היו תא פולשנית 5, בדומה למה שכבר הוכח לAβ 6, 7-9 טאו, α סינוקלאין-10, 11 polyglutamine, או SOD1 אגרגטים 12. ממצאים אלה הניעו אותנו להציע שלפחות משנה של מקרים עם CMD, אלה עם DISC1 המצטבר עלולים להיות הפרעות חלבון קונפורמציה. כאן אנו מתארים כיצד אנו יוצרים DISC1 aggresomes בתאי יונקים, לטהר אותם בשיפוע סוכרוז ולהשתמש בם לStudi תא הפולשנותes. כמו כן, אנו מתארים כיצד אנחנו מייצרים C-מסוף DISC1 בר, תווית באופן בלעדי multimeric ולטהר אותו ללימודי הפולשנות סלולרית. שימוש בmultimers רקומביננטי של DISC1 להשיג הפולשנות תא דומה כמו לבר סינטטי שכותרת דומה α-סינוקלאין. אנו גם מראים שקטע זה נלקח בvivo כאשר מזריקים stereotactically לתוך המוח של בעלי חיים מושתלים.

Watch this Scientific Journal Video about Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species at JoVE.com

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

פלישת הדור, טיהור ותא של aggresomes התאי, cytoplasmic אורך המלא DISC1 חלבון מתרביות תאים ומתויג בר, multimeric רקומביננטי DISC1 חלבון<em&gt; א coli</em&gt; מתוארים. הפולשנות סלולרית מוצגת לתאי נמען בתרבית תאים ונוירונים<em&gt; In vivo</em&gt; לאחר חיסון המוח stereotactical.

דור, טיהור, ואפיון של מיני חלבון DISC1 הסלולריים פולשניים

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

generation-purification-characterization-cell-invasive-disc1-protein

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Biology

13.6K Views.  Medical School Düsseldorf, Germany. פלישת הדור, טיהור ותא של aggresomes התאי, cytoplasmic אורך המלא DISC1 חלבון מתרביות תאים ומתויג בר, multimeric רקומביננטי DISC1 חלבון א coli מתוארים. הפולשנות סלולרית מוצגת לתאי נמען בתרבית תאים ונוירונים In vivo לאחר חיסון המוח stereotactical.

Video: Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

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 ...

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.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

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 ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

שיער פולשנית דגימה טכניקה לקבל DNA באיכות גבוהה מ יונקים קטנים החמקמק

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

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 ...

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 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 ...

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  ...

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 ...

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H2O2 production, respectively.

We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.

איתור, חזותי וquantitating הדור של מינים חמצן תגובתי במערכת מודל אמבה

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

זיהוי אינטראקציה בין חלבונים ב<em&gt; דרוזופילה</em&gt; ראשי מבוגרים טנדם זיקה טיהור (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...