This work was supported by Wellcome Trust grant 088419 to MDB.

1. טיהור של GTPase מתויג GST <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תרבות E. זן coli כגון BL21 הפך עם pGEX-Rac1 O / N ב 37 מעלות צלזיוס, רועד ב 220 סל&quot;ד, 500 מיליליטר של תקשורת autoinduction (25 מ&quot;מ Na 2 HPO 4, 25 מ&quot;מ KH 2 PO 4, 50 מ&quot;מ NH 4 Cl, 5 מ&quot;מ Na 2 SO 4, 2 מ&quot;מ MgSO 4, 2 מ&quot;מ CaCl 2, 0.5% גליצרול, גלוקוז 0.05%, 0.2% לקטוז, 5 גרם Tryptone, תמצית שמרים 2.5 גרם, 100 מיקרוגרם / מיליליטר אמפיצילין). </li><li style=";text-align:right;direction:rtl"> חיידקי קציר על ידי צנטריפוגה במשך 10 דקות ב 10,000 XG, 4 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> Resuspend גלולה חיידקים במגיבים מיצוי חלבון 20 מיליליטר, מעכבי פרוטאז 1x ודגירה של 20 דקות ב RT עם היפוך. </li><li style=";text-align:right;direction:rtl"> להבהיר את lysate ידי צנטריפוגה ב40,000 XG למשך 30 דקות. </li><li style=";text-align:right;direction:rtl"> להוסיף חרוזים מגנטיים 2 מיליליטר גלוטתיון, לשטוף עם פוספט שנאגרו המלוח (PBS: 10 מ&quot;מ Na 2 HPO 4, 1.8 מ&quot;מ KH 2 PO 4, 137 מ&quot;מ NaCl, 2.7 מ&quot;מ KCl). </li><li style=";text-align:right;direction:rtl"> דגירה עבור שעה 2, ערבוב על ידי היפוך על 4 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> לשטוף חרוזים טעונים חלבון ארבע פעמים עם 10 מיליליטר PBS, באמצעות סדרן חלקיקים מגנטי כדי לזרז את החרוזים בכל שלב. </li><li style=";text-align:right;direction:rtl"> Resuspend חלבון טעון חרוזים ב 2 מיליליטר PBS ולאחסן ב -80 ° C ב 100 aliquots μl עד צורך. </li></ol> 2. ביטוי של חלבונים מחייב GTPase <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> היום לפני הניסוי, פלסמידים transfect קידוד חלבון פלואורסצנטי ירוק (GFP) -tagged גרסאות של כל חלבון קושר GTPase ל- 75 סנטימטר 2 בקבוק נפרד של HEK293T כדלקמן. לאימות של טעינת נוקלאוטיד, מתויג GFP transfect TrioD1 ל- 75 סנטימטר 2 בקבוק שלישי של HEK293T. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לדלל polyethylamine עד 1 מ&quot;ג / מיליליטר ב 100 μl סטרילי 150 מ&quot;מ NaCl. </li><li style=";text-align:right;direction:rtl"> להוסיף polyethylamine המדולל 27 μl 223 μl מופחת תקשורת בסרום. </li><li style=";text-align:right;direction:rtl"> להוסיף פלסמיד דנ&quot;א מיקרוגרם 12 עד 250 μl מופחת תקשורת בסרום. </li><li style=";text-align:right;direction:rtl"> דגירה צינור אחד עבור 2 דקות ב RT. </li&gt; <li style=";text-align:right;direction:rtl"> מערבבים את polyethylamine וDNA מתערבב בצינור יחיד ומערבולת למשך 2 דקות. </li><li style=";text-align:right;direction:rtl"> דגירה של 15-20 דקות ב RT. </li><li style=";text-align:right;direction:rtl"> החלף את המדיה הצמיחה (נשר מדיה השתנה Dulbecco, 10% בסרום שור עוברי, L-גלוטמין 2 מ&quot;מ, ללא אנטיביוטיקה) על 90% HEK293T מחוברות עם 5 מיליליטר תקשורת צמיחה טרי. </li><li style=";text-align:right;direction:rtl"> מוסיף את תערובת polyethylamine / DNA המשולב לבקבוק ודגירת O / N ב 37 מעלות צלזיוס, 5% CO 2. </li></ol></li></ol> 3. טיהור של חלבונים מחייב GTPase <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> יש לשטוף את צלוחיות של תאי transfected בPBS ולנקז בקבוק במשך 5 דקות, aspirating נוזל חופשי. </li><li style=";text-align:right;direction:rtl"> לגרד את תאים ב500 חיץ תמוגה μl (50 מ&quot;מ טריס-HCl (pH7.8), 1% Nonidet P-40, 1x מעכבי פרוטאז) לצינור microfuge. </li><li style=";text-align:right;direction:rtl"> תאי Lyse על ידי ערבוב על ידי היפוך על 4 מעלות צלזיוס למשך 30 דקות. </li><li style=";text-align:right;direction:rtl"> במהלך תמוגה, לשטוף שני המון חרוזים GFP-מלכודת 40 μl שלוש פעמים עם חיץ תמוגה טרי, חרוזים סדימנטציה ב2,700 XG למשך 2 דקות בין שוטף. </li><li style=";text-align:right;direction:rtl"> להבהיר lysates על ידי צנטריפוגה ב21,000 XG במשך 10 דקות. </li><li style=";text-align:right;direction:rtl"> העברה הבהירה lysate של כל אחד מחלבוני המתחרה להפריד חרוזים GFP-מלכודת שטפו ולאפשר חלבוני GFP-היתוך להיקשר לשעה 2, ערבוב על ידי היפוך על 4 מעלות צלזיוס. שמור lysate מתאי ה- GFP-TrioD1 על קרח. </li><li style=";text-align:right;direction:rtl"> לשטוף חרוזים GFP-מלכודת נטענים פעמיים ב 50 מ&quot;מ טריס-HCl (pH 7.8), 50 מ&quot;מ NaCl, 0.7% (w / v) Nonidet P-40 ופעמים ב 50 מ&quot;מ טריס-HCl (pH 7.6), 20 מ&quot;מ MgCl 2 , סדימנטציה חרוזים ב2,700 XG למשך 2 דקות בין שוטף. </li><li style=";text-align:right;direction:rtl"> Elute חלבוני GFP-היתוך על ידי הוספת 40 μl 0.2 M גליצין (pH 2.5) וpipetting למעלה ולמטה במשך 30 שניות. מייד חרוזים משקעים ב21,000 XG במשך 60 של ונוזל להעביר צינור microfuge חדש המכיל 4 μl 1 M טריס- HCl (pH 10.4). לעשות את זה מהר כדי לצמצם את הפגיעה בחלבון המטוהר. </li><li style=";text-align:right;direction:rtl"> לנתח 1 μl של כל חלבון מטוהר על ידי כתם מערבי ובדיקה עם נוגדן אנטי GFP להקים תשואה היחסית באמצעות blott כמותייםing מערכת על פי הפרוטוקול של היצרן. לחלופין, לקבוע ריכוזי חלבון על ידי חומצת assay bicinchoninic (BCA), אבל זה מציג שגיאות אם החלבונים לא מגיבים עם assay באופן זהה או יש חלבונים מזהמים. </li><li style=";text-align:right;direction:rtl"> להשוות ריכוז חלבון טוחנת על ידי התוספת של 50 מ&quot;מ טריס-HCl (pH 7.6), 20 מ&quot;מ MgCl 2. </li></ol> 4. טעינת נוקלאוטיד של GTPase <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להפשיר aliquot אחד חרוזים מגנטיים GST-Rac1, מוכנים בשלב 1. </li><li style=";text-align:right;direction:rtl"> קח 90 μl של חרוזים GST-Rac1 ולשטוף שלוש פעמים עם 20 מ&quot;מ טריס- HCl (pH 7.6), 25 מ&quot;מ NaCl, 0.1 מ&quot;מ DTT, 4 מ&quot;מ EDTA, באמצעות סדרן חלקיקים מגנטי כדי לזרז את החרוזים בכל שלב. </li><li style=";text-align:right;direction:rtl"> מאגר לשאוב מחרוזים ולהוסיף 100 μl 20 מ&quot;מ טריס- HCl (pH 7.6), 25 מ&quot;מ NaCl, 0.1 מ&quot;מ DTT, 4 מ&quot;מ EDTA. </li><li style=";text-align:right;direction:rtl"> לדברי האם התמ&quot;ג, GTP או לא טעינת נוקלאוטיד נדרש לניסוי התחרות, להוסיף 12 μl 100 מ&quot;מ תמ&quot;ג, μl 12 10 מ&quot;מ ג.א.anosine 5 &#39;- [γ-thio] אדנוזין (GTPγS) או לא נוקלאוטיד לחרוזי GST-Rac1 60 μl. </li><li style=";text-align:right;direction:rtl"> לבקרות נוקלאוטיד-הטעינה, לפצל את חרוזים שנותרו לשלושה aliquots 10-μl ולהוסיף 2 μl 100 מ&quot;מ תמ&quot;ג, 2 μl 10 מ&quot;מ GTPγS או לא נוקלאוטיד על צינור אחד. </li><li style=";text-align:right;direction:rtl"> דגירה תערובות חרוז למשך 30 דקות ב 30 מעלות צלזיוס עם תסיסה. </li><li style=";text-align:right;direction:rtl"> לייצב Rac1 מאוגד נוקלאוטיד על ידי תוספת של 1 M MgCl 2: 3 μl לתערובת הניסיונית (שלב 4.4), 0.5 μl לכל אחד מתערובות השליטה (שלב 4.5). </li></ol> 5. תחרות המחייבת. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הגדרה 6 צינורות microfuge, המכיל: 200 μl 50 מ&quot;מ טריס-HCl (pH 7.6), 20 מ&quot;מ MgCl 2 חרוזים 10 μl ניסיוניים טעון נוקלאוטיד Rac1 (משלב 4.7) חלבון 5 μl Rac1 מחייב (חלבון מחייב קבוע) </li><li style=";text-align:right;direction:rtl"> על צינור אחד, להוסיף חלבון B 0, 1, 2.5, 5, 10 או 20 μl מחייב Rac1 (חלבון מחייב משתנה). כרכים אלה מניחיםpproximately ריכוזי המניה שווים של חלבונים המחייבים קבועים ומשתנים וייתכן שיצטרכו להיות מותאמים. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> התאם כרכים של חלבונים מחייבים A ו- B אם יש הבדלים גדולים בזיקות המחייבות של שני חלבונים ואת זה צריך להיקבע באופן אמפירי באמצעות החזרות הניסיוניות. מרכיבים את הנפח הכולל של התערובת מחייב 235 μl על ידי תוספת של 50 מ&quot;מ טריס-HCl (pH 7.6), 20 מ&quot;מ MgCl 2. </li></ol></li><li style=";text-align:right;direction:rtl"> הגדר את צינור microfuge המכיל: 200 μl 50 מ&quot;מ טריס-HCl (pH 7.6), 20 מ&quot;מ MgCl 2 חרוזים 10 μl ניסיוניים טעון נוקלאוטיד Rac1 (משלב 4.7) חלבון 10 μl Rac1 מחייב (חלבון מחייב קבוע) </li><li style=";text-align:right;direction:rtl"> הגדרת התמ&quot;ג, GTPγS ולא צינורות שליטת נוקלאוטיד: 200 μl 50 מ&quot;מ טריס-HCl (pH 7.6), 20 מ&quot;מ MgCl 2 10 חרוזים Rac1 שליטת μl נטענים בשלב 4.5 עם תוצר, GTPγS או לא נוקלאוטיד והתייצבו בשלב 4.7. 180 μl Hlysate EK293T GFP-TrioD1, מוכן כמו בשלב 3.6 4 μl 1 M MgCl 2 </li><li style=";text-align:right;direction:rtl"> דגירה את התערובת לשעה 2, ערבוב על ידי היפוך על 4 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> שטוף את החרוזים שלוש פעמים עם 50 מ&quot;מ טריס-HCl (pH 7.6), 20 מ&quot;מ MgCl 2. </li><li style=";text-align:right;direction:rtl"> Elute מחויב חלבונים בμl 20 הפחתת חיץ מדגם (50 מ&quot;מ טריס-HCl (pH 7), 5% SDS, גליצרול 20%, 0.02 מ&quot;ג / מיליליטר bromophenol, 5% β-mercaptoethanol הכחול). </li></ol> 6. ניתוח של תחרות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לפתור 10 μl של החלבון הקשור (שלב 5.6) על ידי ג&#39;ל אלקטרופורזה polyacrylamide סולפט dodecyl נתרן (SDS-PAGE) וכתם מערבי. </li><li style=";text-align:right;direction:rtl"> דגירה הקרום על 4 מעלות CO / N מדולל 1/1000 בנוגדן אנטי-GFP בחסימת המאגר מדולל ל1x PBS, 0.1% Tween-20 לזהות שניהם מחייב חלבוני GTPase מתויגים. </li><li style=";text-align:right;direction:rtl"> לשטוף את הממברנה שלוש פעמים במשך 10 דקות עם PBS, 0.1% Tween-20. </li><li style=";text-align:right;direction:rtl"> דגירה הממברנה במשך 30 דקות ב RT בDyLight שניות נגד ארנב 800 מצומדות-נוגדן ondary, בדילול מלא 1 / 10,000 בחסימת המאגר מדולל ל1x PBS, Tween-20 0.1%. </li><li style=";text-align:right;direction:rtl"> לשטוף את הממברנה שלוש פעמים במשך 10 דקות עם PBS, 0.1% Tween-20. </li><li style=";text-align:right;direction:rtl"> סרוק את הקרום באמצעות מערכת הדמיה אינפרא אדום, שימוש בתוכנה למדידת עוצמת להקה על פי הפרוטוקול של היצרן. </li><li style=";text-align:right;direction:rtl"> עלילה עוצמת הלהקה של כל חלבון נגד נפח של המתחרה משתנה (חלבון B). </li><li style=";text-align:right;direction:rtl"> מחלקים את הנפח של מתחרה משתנה בנקודה שבה הקווים מצטלבים בנפח של מתחרה קבועה (חלבון, 5 μl) כדי לקבוע את יחס המתחרה שבשיווי המשקל מושגת. </li><li style=";text-align:right;direction:rtl"> לאימות מעמד נוקלאוטיד-טעינה, קרומי בדיקה לקינאז מופעל p21 1 (PAK1) (מפעיל) וה- GFP-TrioD1 (GEF), כמתואר בצעדים 6.1-6.6. </li></ol>

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יש המחברים אין לחשוף.

This protocol describes a method for comparing the relative affinities of pairs of small GTPase-binding proteins. The key steps are the preparation of purified GTPase-binding proteins and the nucleotide loading of the GTPase. The use of GTPase-binding proteins with the same GFP tag, allows the concentrations at which similar amounts of each competitor binds to be accurately determined. The use of recombinant nucleotide-loaded GTPase allows interrogation of the binding properties of the GTPase under specific activity conditions. This step is also the most sensitive as nucleotides will both hydrolyze and detach from the GTPase if the magnesium conditions are not maintained precisely.
In the cell, the large number of GTPase-binding proteins combined with the rapid nucleotide turnover makes such pathways difficult to interpret. The simplicity of this method in comparing only pairs of binding proteins and using carefully controlled nucleotide-loading conditions allows signaling pathways to be elucidated. However, the greatest strength of the protocol is also the greatest weakness as it is a simplification of the in vivo situation. Competition assays can be used to build a robust hypothesis, but this should then be tested in cells by knockdown experiments.
There are three features that must be considered when selecting the GFP-tagged GTPase-binding proteins to be used in the experiment. First, the fusion proteins must express well in mammalian cells, such as HEK293T, as competition assays require a reasonable amount of protein. Second, it must be possible to purify the recombinant protein without significant degradation, and where this is not possible, cloning of a GTPase-binding fragment should be considered. Third, the two GTPase-binding proteins must resolve from one another on SDS-PAGE to allow analysis in section 6.
There are a number of potential caveats to the experiment that need to be considered, and possibly addressed:
Possible denaturation of purified GTPase-binding proteins during the acid elution step or steric hindrance by the GFP tag. In our hands, these have not been a problem, but must be tested. The purified proteins can be tested in functional assays 10. Commercial kits now exist for testing the activity of GEFs or GAPs without the need for isotope-labeled nucleotides. Sequestering proteins, by their nature protect GTPases from GEF or GAP activity, so can be used as competitive inhibitors in the commercial GEF or GAP assays, as we did in our recent publication 7. The relevant feature of proteins that traffic GTPase are the capacity to bind the GTPase, and this can be tested easily in a pull down assay. An alternative approach to testing protein integrity that is applicable to all binding proteins is to titrate protein eluted from GFP-trap beads with glycine with the same protein removed from GFP-trap beads by enzymatic cleavage. The experiment would be analyzed by probing both the GFP-tagged and cleaved protein with an antibody against the protein itself. If the protein is undamaged by elution, equilibrium should be achieved at a 1:1 ratio. This approach would also indicate whether the presence of the GFP tag itself compromises the binding properties of the candidate protein, though this does require the production of a construct with an enzymatic cleavage site between the tag and the binding protein. Whether the protein is compromised by the tag or the elution step, the problem could be addressed by modifying the protocol to use an alternative purification method. Rather than GFP, binding proteins could be His-tagged, purified using Ni-NTA and analyzed using an antibody against the His-tag. The important feature is that both binding proteins must share a common tag although, if necessary, two tags could be added to a protein, one for purification and the other for detection.
The protocol is designed to investigate competition between interactions with the switch I/II domains. Although the majority of GTPase interactions are mediated by this motif, there are some exceptions, most notably the interactions of GDIs that bind to the prenyl tail, as well as obscuring the switch domains. In principle, the protocol could be adapted to use GTPase purified from mammalian cells, so that the GTPase is prenylated, however, the presence of multiple binding sites or allosteric effects complicate the interpretation of competition-binding data. Further problems associated with such a modification are that GDIs co-purify with GTPase from mammalian cells, compromising the purity of the isolated proteins and the hydrophobic nature of the prenyl groups means that prenylated GTPases are associated with either GDI or lipid membrane and such factors would need to be considered in the experiment.
The amount of GST-Rac1 being used in the assay. The constant GTPase binding protein must be at a greater concentration than the Rac1, or when the competitor is added, it will simply bind to free Rac1. It will be immediately obvious if this has happened as binding of the competitor, without a loss of the constant protein, will be detected in much the same way as when the two competing proteins bind to one another as shown in Figure 3B. As an additional control (Step 5.3), a binding reaction containing double the amount of constant binding protein and no variable binding protein should be included (Step 5.3). If the Rac1 in the titration experiment is saturated, doubling the amount of constant binding protein will have no effect on the output. The volumes suggested in the protocol should be appropriate, but the amount of Rac1 can be easily reduced. If binding of the competitor without loss of the constant binding partner is observed, reducing the amount of Rac1 should be attempted before trying to map binding sites to avoid ternary complex formation.
Non-specific interaction of GTPase-binding proteins with the GST or bead, as well as specifically with Rac1. This problem would be manifested by residual binding of the constant GTPase-binding protein, even when the variable GTPase-binding protein has reached a plateau at high concentration. Identification of this issue will be aided by conducting reciprocal experiments where the constant and variable GTPase-binding proteins are swapped. Reciprocal experiments will also greatly improve the accuracy of the estimate of equilibrium point, so should always be included. In cases of non-specific binding, the relative concentrations at which equilibrium is achieved can still be calculated by comparing band intensity between the maxima and minima for each protein, or by measuring the extent of non-specific binding by using GST beads as bait, rather than GST-Rac1.
Pull down assays using different nucleotide-loading conditions should be used to complement the competition assay described in this protocol. Determining the nucleotide preference of partners is important for both understanding the competition events and understanding the signaling pathway that the GTPase-binding protein is involved in. In Figure 2B we analyze binding of proteins with established preference for GTP-loaded or nucleotide-free GTPase as a means to validate nucleotide loading. However, it is sensible to investigate the effect of nucleotide loading on each of the competitors as well. If the hypothetical competitors show different preferences, competition will make less of a contribution to the signaling pathway, and indeed nucleotide turnover is likely to be the mechanism that directs exchange of the binding proteins.

The actin cytoskeleton that determines the shape, polarity and migratory properties of mammalian cells is regulated by the Rho-family of small GTPases. The Rho-family GTPases include RhoA that stimulates cytoskeletal contraction, Rac1 that stimulates actin branching and membrane protrusion, and Cdc42 that has similar effects on actin polymerization to Rac1 and causes the formation of filopodia 1,2. GTPase signaling activity is determined by binding of a nucleotide, which controls the contraction and relaxation of the switch I and switch II loops that mediate the protein-protein interactions with both regulators and effectors. Guanosine 5&#8217;-triphosphate (GTP)-bound GTPases activate downstream effectors, whereas the Guanosine 5&#8217;-diphosphate (GDP)-bound form is inactive. In the cell, cycles of GTP hydrolysis and nucleotide exchange allow rapid turnover of GTPase signals that are necessary for cytoskeletal dynamics. Nucleotide turnover is regulated by three mechanisms. Guanine nucleotide exchange factors (GEFs) stabilize the nucleotide-free GTPase, catalyzing exchange of GDP for GTP, and thereby stimulating GTPase signaling activity 3,4. GTPase-activating proteins (GAPs) catalyze hydrolysis of GTP to GDP, thereby inhibiting GTPase signaling activity 5. Sequestering molecules such as regulator of chromatin condensation 2 (RCC2) and guanine nucleotide dissociation inhibitors (GDIs) obscure the switch loops and in the case of GDIs remove the GTPase from the membrane by interaction with the prenyl tail 6,7. Each of the three classes of regulatory molecule interact with the switch loops, as do the downstream effectors and some trafficking regulators such as coronin-1C 7. The purpose of this protocol is to measure competition for the switch I/II binding site between putative regulators and downstream signaling molecules. It should be noted that competition assays test binding to a shared binding site, so that this protocol is not suitable for testing interactions with other sites, such as binding of GDIs to the prenyl tail.
The subtlety of the conformation differences between active and inactive forms, combined with the labile nature of the bound nucleotide, has made study of GTPase-binding events difficult. The role of the bound nucleotide means that conventional binding assays such as immunoprecipitation or surface plasmon resonance are not well suited to investigation, as the nucleotide cannot be controlled. This obstacle is compounded by the overlap in the binding sites of GEFs, GAPs, effectors, sequestering molecules and trafficking molecules, which make binding data for a single interaction difficult to interpret in the context of the competition that will occur in the cell. Immunoprecipitation, in particular, is compromised by competition between binding partners, as under certain cellular conditions, one binding partner might be identified at the expense of all others, while under other conditions, another partner might dominate. The dynamic nature of GTPase signaling is essential to GTPase function and must be considered when analyzing the relationships between the binding interactions of different regulators. Indeed, we recently described a pathway that relied heavily on competitive binding. We identified coronin-1C as a trafficking molecule that bound to the switch loops of GDP-Rac1 7. In areas of low GEF activity, trafficking would dominate, removing Rac1 from those regions. However, when Rac1 is delivered to regions of the cell where GEF activity is high, the GEF would outcompete coronin-1C, thereby both activating Rac1 and preventing coronin-1C-mediated removal of Rac1 from that area. The model goes further, because the action of the GEF exchanges bound GDP for GTP, shifting the equilibrium still further from coronin-1C. Consequently, Rac1 activity could be explained entirely in terms of competition and relative affinity.
In this protocol, we describe a method for comparing the relative affinities of different binding partners for small GTPases, using Rac1 as an example. By using a purified protein approach, it is possible to piece together a chain of signaling events by pair wise comparison, in an experiment where the bound nucleotide can be closely controlled.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Bugbuster</td>
			<td>Novagen</td>
			<td>70584-3</td>
			<td></td>
		</tr>
		<tr>
			<td>COMPLETE protease inhibitor</td>
			<td>Roche</td>
			<td>05 056 489 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione magnetic beads</td>
			<td>Pierce</td>
			<td align="right">88821</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine, branched, average Mw ~25,000</td>
			<td>Sigma Aldrich</td>
			<td>408727-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>OPIMEM</td>
			<td>Life Technologies</td>
			<td>31985-047</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Media</td>
			<td>Sigma Aldrich</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>10270-1-6</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Life Technologies</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP-Trap_A</td>
			<td>Chromotec</td>
			<td>gta-20</td>
			<td></td>
		</tr>
		<tr>
			<td>GDP</td>
			<td>Sigma Aldrich</td>
			<td>G7127</td>
			<td>Highly unstable. Aliquot and store at -80 immediately upon reconstritution</td>
		</tr>
		<tr>
			<td>GTP&#947;S</td>
			<td>Sigma Aldrich</td>
			<td>G8634</td>
			<td>Highly unstable. Aliquot and store at -80 immediately upon reconstritution</td>
		</tr>
		<tr>
			<td>Blocking Buffer</td>
			<td>Sigma Aldrich</td>
			<td>B6429</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma Aldrich</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP antibody</td>
			<td>Living Colors</td>
			<td align="right">632592</td>
			<td>Use at 1/1000 dilution</td>
		</tr>
		<tr>
			<td>DyLight 800 conjugated goat anti-rabbit secondary antibody</td>
			<td>Fisher Scientific</td>
			<td align="right">10733944</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PAK1 antibody</td>
			<td>Cell Signaling</td>
			<td>2602S</td>
			<td>Use at 1/1000 dilution</td>
		</tr>
		<tr>
			<td>Odyssey SA Infrared Imaging System</td>
			<td>Li-cor</td>
			<td>9260-11PC</td>
			<td></td>
		</tr>
	</tbody>
</table>

comparing the affinity of gtpase-binding proteins using competition assays

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the binding capabilities of GTPases for two reasons: The fact that all interactions involve the same face of the GTPases means that binding events must be considered in the context of competitors, and the fact that the bound nucleotide must also be controlled means that conventional approaches such as immunoprecipitation are unsuitable for GTPase biochemistry. The assay relies on the use of purified proteins. Purified Rac1 immobilized on beads is used as the bait protein, and can be loaded with GDP, a non-hydrolyzable version of GTP or left nucleotide free, so that the signaling stage to be investigated can be controlled. The binding proteins to be investigated are purified from mammalian cells, to allow correct folding, by means of a GFP tag. Use of the same tag on both proteins is important because not only does it allow rapid purification and elution, but also allows detection of both competitors with the same antibody during elution. This means that the relative amounts of the two bound proteins can be determined accurately.

<html> פרוטוקול זה נועד לחשב את הזיקות היחסית של שותפים מחייבים Rac1, ללא הצורך לדעת את הריכוז המדויק של המתחרים (איור 1). קביעת ריכוז חלבון מציגה שגיאות וכאשר בוחנת את תחרות בין מולקולות במסלול איתות אינו נחוצה. עם זאת, חשוב לדעת כי יש שני מתחרים באותו ריכוז טוחנת בפתרונות המניות כדי לאפשר יחסים פשוטים שיחושבו בעת הוספת נפחים שונים לassay. יש לי 40 μl של חרוזים GFP-מלכודת יכולת מחייבת של ~ 300 pmol כך מחוברות 75 סנטימטר 2 צלוחיות של מאוד לבטא בתאים יהיה להרוות את חרוזים, והתוצאה הוא שההכנות של שני חלבונים מחייבים השונים תהיה דומות לפני ההתאמה (איור 2 א). אם אחד מהחלבונים מבטא בצורה גרועה, בעיה זו ניתן להתגבר על ידי טיהור חלבון שמן בקבוק אחד או יותר של תאים. ss = &quot;jove_content&quot;&gt; המחייב של רוב effectors GTPase ורגולטורים תלוי בנוקלאוטיד-הטעינה של GTPase הפיתיון, ולכן חשוב לבדוק אם הטעינה הייתה מוצלחת. טעינה יכולה להיות מאומת על ידי מזרז חלבונים מחייבים ידועים מlysates התא. חלבוני מפעיל, כגון לאגד PAK1 לGTP-Rac1 וניתן זירז בקלות מlysates וזוהו על ידי מערבי סופג 8 (איור 2). GEFs להיקשר מעדיף לנוקלאוטיד ללא GTPase לייצב את מצב המעבר. כGEFs הוא של שפע נמוך, בדרך כלל לא פעיל ולעתים קרובות למחוק גרוע, עדיף לביטוי יתר GEF או בר GEF לGTPase ללא נוקלאוטיד מבחן. אנו משתמשים לעתים קרובות ההומולוגיה, הזוגית הראשונה של שלישייה, הביע כהיתוך GFP (GFP-TrioD1 9) (איור 2), אך כל GEF יעבוד. חלבונים שנקשרים לGTPase טעון התמ&quot;ג הם נדירים יותר. לאחרונה דיווחו RCC2 כחלבון אחד כזה 7, או תמ&quot;ג טעינה יכולה להיות מאומת פשוט כמו בינדיng ללא GEF ולא מפעיל. הפלט מהניסוי יהיה כתם מערבי המתאר את שני שותפים מחייבים מתויג GFP חייבים GTPase. על ידי שימוש בנוגדנים חד לזהות שני החלבונים, ניתן לקבוע את הריכוזים שבי כמויות דומות של שני המתחרים לאגד ולכן הזיקות יחסי להסיק. בדוגמא זו תחרות בין תחום המדחף של חלבון Rac1-הסחר, coronin-1C (Rac1 מחייב חלבון), וחלבון Rac1-sequestering, RCC2 (Rac1 מחייב חלבון B), הוא הוכיחו (איור 3 א). על ידי שימוש בנפח קבוע של מדחף coronin-1C (5 μl), והוספת כמויות הולכות וגדל של RCC2, אנו יכולים לראות מGFP למחוק הוא הגיע לשיווי המשקל שב1.25-2.5 μl של RCC2 (כוכבית), הוכחה כי RCC2 יש חזק זיקה לRac1 מ coronin-1C. על ידי מדידת עוצמת להקות באמצעות מערבי סופג כמותי, וזומם ערכים ממוצע לכל competitor, נקודת שיווי המשקל יכולה להיות מחושבת בצורה מדויקת על ידי זיהוי הכרכים שבעקומות מצטלבות (איור 3). אחד המכשולים האפשריים לassay תחרות מוצלח הוא אם השותפים מחייבים לקשור זה לזה, כמו גם מחייב Rac1. ב+ 3 א B איור אנחנו מדגימים תחרות בין RCC2 ותחום המדחף של coronin-1C, ולא coronin-1C באורך מלא. הסיבה לשימוש בcoronin הקטוע היא שcoronin-1C גם נקשר RCC2 דרך תחום הזנב. כאשר אורך מלא coronin-1C הוא טיטרציה נגד RCC2, כריכה של שני החלבונים מזוהה, עקב היווצרות משולשת מורכבת, ולא תחרות (איור 3 ג). אם תחרות מתרחשת, הכריכה של חלבון אחד תגדל ואילו ירידות האחרות, וסך מחויב GFP-ההיתוך יישאר קבוע. במקרים בהם מורכב משולש יוצר יש צורך לחתוך אחד של החלבון קושר GTPase כך שcompetitors אינטראקציה כבר לא. <img alt="איור 1" src="/files/ftp_upload/53254/53254fig1.jpg" /> איור 1. זרימת עבודה. ייצוג סכמטי של זרימת העבודה לקביעת הזיקה של חלבונים מחייב GTPase באמצעות מבחני תחרות. <a href="https://www.jove.com/files/ftp_upload/53254/53254fig1large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> <img alt="איור 2" src="/files/ftp_upload/53254/53254fig2.jpg" /> איור 2. אימות של חלבונים מטוהרים. () מטוהרים Rac1 מחייב חלבונים נותחו על ידי כתם מערבי, חיטוט באנטי-GFP כדי לקבוע את התשואה היחסית של שני החלבונים מתויג GFP. סוג זה של השוואה במהלך הניסוי מאפשר הריכוז של שני החלבונים להיות מותאם כך שיתאימו בניסוי המחייב. (ב) GDP, GTPγS ולא GST-Rac1 טעון נוקלאוטיד הודגר עם lysate מHEK293T להביע GFP-TrioD1 ומחויב חלבונים זוהו על ידי קשירת קשר לPAK1 אנדוגני או ביטוי יתר GFP-TrioD1. <a href="https://www.jove.com/files/ftp_upload/53254/53254fig2large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> <img alt="איור 3" src="/files/ftp_upload/53254/53254fig3.jpg" /> איור 3. ניתוח כתם מערבי של חלבון ביחס מחייב. תפוקות דוגמא ממבחנים מחייב תחרות. טעון תמ&quot;ג () Rac1 היה מעורב עם תחום מדחף 5 μl GFP-coronin-1C והגדלת נפחים של ה- GFP-RCC2 היו טיטרציה ב. ב חלבונים קשורים מערבי סופג לGFP, נושאים עם זיהוי ההפרש של שני החלבונים הם נמנעו ואות ה- GFP מדווחת יחס טוחנת בין שני חלבוני האיחוי. כוכביות לציין את יחסי תחרות על eitheצד r של נקודת שיווי המשקל. (ב) בעוצמות בנד של חלבוני היתוך GFP קשורים משלושה ניסויים בלתי תלויים נמדדו על ידי מערבי סופג כמותי, באמצעות נוגדנים משניים fluorophore מצומדות וממוצעים זממו כדי לחשב את הסכום של RCC2 הדרושים כדי להגיע לשיווי משקל. (C ) פלט דוגמא מניסוי שבו חלבוני Rac1 מחייבים להיקשר זה לזה ויוצרים מורכב משולש, ולא מתחרה. Rac1 היה מעורב עם GFP-RCC2 5 μl והגדלת נפחים של ה- GFP-coronin-1C באורך מלא טעון תמ&quot;ג היה טיטרציה ב. הגידול בGFP-coronin-1C כבול ללא אובדן של ה- GFP-RCC2 כבול מציינת היווצרות מורכבת משולשת. <a href="https://www.jove.com/files/ftp_upload/53254/53254fig3large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a>

Watch this Scientific Journal Video about Comparing the Affinity of GTPase-binding Proteins using Competition Assays at JoVE.com

Comparing the Affinity of GTPase-binding Proteins using Competition Assays

This protocol compares the relative affinities of binding partners for Rho-family GTPases, including Rac1. In vivo, Rac1-binding proteins compete for a single binding interface, the conformation of which is dictated by a bound nucleotide. The nucleotide is both important and difficult to control experimentally, due to the high hydrolysis rate.

השוואת הזיקה של חלבונים מחייב GTPase באמצעות מבחני תחרות

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the ...

comparing-affinity-gtpase-binding-proteins-using-competition

Research

JoVE Journal

Biology

9.0K Views.  University of Bristol. This protocol compares the relative affinities of binding partners for Rho-family GTPases, including Rac1. In vivo, Rac1-binding proteins compete for a single binding interface, the conformation of which is dictated by a bound nucleotide. The nucleotide is both important and difficult to control experimentally, due to the high hydrolysis rate.

Video: Comparing the Affinity of GTPase-binding Proteins using Competition Assays

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

ניתוח של שוואן-astrocyte אינטראקציות שימוש במבחנה מבחני

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

התגבשות של חלבונים בממברנה ב Mesophases Lipidic

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

תפוקה גבוהה התגבשות של חלבונים בממברנה שימוש בשיטה Lipidic Bicelle

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

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 ...

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. ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

במבחני צבירת מבחנה באמצעות חלבון Hyperphosphorylated טאו

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

חוקר את החלבונים כמו פריון-הפצת ורעילות של שימוש במטזואניים דגם אורגניזם<em&gt; C. elegans</em

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

זיהוי של Myod Interactome שימוש טיהור טנדם זיקה מצמידים ספקטרומטריית מסה

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...