ࡱ> 9;8@ #bjbjFF &,,N LLLL`!nccc  $,#R~%!]cc!x!lll l llN | P LFI 0 !0!y 0.&j.&` .& 8c>,l$ccc!!DH b H Preparation of GST-RBD (for RhoA assays) 1) Growth of bacteria prepare ON culture of pGEX GST-RBD (20-40 mL LB with 100 g/mL amp) dilute ON culture 1:50 into a final volume of 1000 mL LB with 100 g/mL amp grow culture to OD of 0.6 induce with 1 mM IPTG ~16 h @ 18 (OD600 nm should be roughly 1.8) 2) Purification of GST-RBD on Glutathione Sepharose 4B pellet bacteria by centrifugation @ 4000 rpm for 10 minutes @ 4C resuspend pellet in 20 mL lysis buffer: 50 mM Tris, pH 7.4 50 mM NaCl 5 mM MgCl2 1 mM DTT protease inhibitors 1 % Triton X-100 lyse bacteria using emulsiflex spin @ 40,000 rpm @ 4C for 30 minutes rotate supernatant for 1 hour with 300-400 l of a 50% slurry of Glutathione Sepharose 4B (wash beads in lysis buffer before adding to supernatant) wash beads 3X in lysis buffer wash beads 3X in lysis buffer with no Triton X-100 store @ 4C for up to 1 week or in 50% glycerol @ -70C 3) analysis of GST-RBD product measure the protein concentration of the beads (should be 3-6 mg/mL) run fusion protein on SDS-PAGE gel and stain with commassie (fusion protein should run around 33 kDa) RhoA Activity Assays Day prior to transfection: split 293T cells 1:2 (do not culture 293 cells for more than a week Transfect P100 dishes of 293 cells with 1 (g of DNA per plate using Lipofectamine 2000 via a standard protocol 24 hours post transfection wash cells 1X with PBS lyse cells in 300 L of Buffer A 50 mM Tris, pH 7.6 500 mM NaCl 0.1 % SDS 0.5 % Deoxycholate 1% Triton X-100 20 mM MgCl2 + inhibitors (1 mM PMSF, 10 g/ml leupeptin, 10 g/ml aprotinin) If lysates become viscous, sonicate for 10 seconds spin for 10 minutes @ 14,000 rpm @ 4C Measure protein concentration via Bradford assay. Dilute lysates with lysis buffer such that each sample contains 1 mg total protein (in 1 mL). Remove 50 microliters of the concentration-equalized lysat*   H n   z+3,0hv>@RTVpǿǿǴǬǿUh1OJQJhOJQJ jmhwOJQJhVW*OJQJhwOJQJhSar5>*OJQJhSar>*OJQJhSarH*OJQJhZOJQJhSarOJQJhSar@)*@- d  . D p  !!!!!!!!!!!!!!!!! & F h^ & F h^ & F h^` & F h^ & F h^# ^&p4HVbw!!!!!!!!!!!!! & F h^` & F h^$`a$gdVW* & F h^ & F h^h@jB\x .!z!!!h#j##!!!!!b0!gdw & F & F h^ ^` & F h^es for lysate loading controls. add 60-80 (g GST-RBD incubate @ 4C with rotation for 60 minutes wash beads 2X in Buffer B (1.5 mL each wash) 25 mM Tris, pH 7.6 40 mM NaCl 30 mM MgCl2 + inhibitors (PMSF, leupeptin, aprotinin) carefully aspirate off supernatant suspend beads in 15 L of SDS-PAGE sample buffer, boil 5 minutes run entire sample (including beads) on 15% SDS-PAGE gel, transfer to PVDF* block 5-15 minutes in TBS-T + 5% milk blot GST-RBD pull-downs with anti-RhoA (1:250) from Santa Cruz. PVDF must be prepared before transferring. This is done by (1) washing with methanol 15 sec., (2) rehydrating with water 1 min., (3) soaking in transfer buffer for 2 min. or longer For loading controls, load 15 microliters of lysate.  BJ\dtv2 < ####hSarH*OJQJhSarOJQJhwOJQJhwhSarOJQJhwhwOJQJhw>*OJQJ/ =!"#$%D@D NormalCJOJQJ_HmH sH tH DAD Default Paragraph FontViV  Table Normal :V 44 la (k(No List >*> Endnote ReferenceH*8+8  Endnote TextCJ:>@: Title$a$5>*OJQJN &)*@-d/Nu ([]^tuCw>q Lo! a b   P 000 0 0 0 00 0 0000000 0 0  0 0 0 00 0 00 00 0 0 0 00000000 0 0 0 0 0 000 0p0 0 0 0 0 00 000 )*@-d/Nu ([]^tuw>q Lo! a b   P M90M90M90M90M90O90O90M90M90O90M90M90M90M90O90M90O90O90M90M90M90M90M90M90M90O900M90M90O90M90M90M90M90M90M90O90M90M90O90O90O90O90O90O90O90O90M90M90O90O90M90O90M90M90M90O900#  # # 8@0(  B S  ?Y vZv[q\TxU U P _ _ P 8*urn:schemas-microsoft-com:office:smarttagsCity9*urn:schemas-microsoft-com:office:smarttagsplace8*urn:schemas-microsoft-com:office:smarttagstime 1350HourMinuteVZ/3CM)2%w{(3<Ya4?6?AJ 7 B P  $.! % P 33333*@u_u"P P  l?]lR>l/l9l; l{0lI1le(;l2=m=l\XBl41Klwk\l ^l&als4;bɔV=f yghl#nlF ul(xlhh^h`CJOJQJo(vhh^h`CJOJQJo(vhh^h`CJOJQJo(vhh^h`CJOJQJo(vhh^h`CJOJQJo(vhh^h`CJOJQJo(vhh^h`CJOJQJo(vhh^h`CJOJQJo(vhh^h`CJOJQJo(vhh^h`CJOJQJo(vhh^h`CJOJQJo(vhh^h`CJOJQJo(vhh^h`CJOJQJo(vhh^h`CJOJQJo(vhh^h`CJOJQJo(v^`OJPJQJo( ^`OJQJo(o pp^p`OJQJo( @ @ ^@ `OJQJo( ^`OJQJo(o ^`OJQJo( ^`OJQJo( ^`OJQJo(o PP^P`OJQJo( hh^h`OJQJo(hh^h`CJOJQJo(vhh^h`CJOJQJo(vhh^h`CJOJQJo(vhh^h`CJOJQJo(vV=f; I19\XBygh?]{0/(x ^2=m=&a41Kwk\F u#nR> e(;s4;bwVW*NyYSar1ZM P 1@xXddddddN P@P P@PP @PPUnknownGz Times New Roman5Symbol3& z Arial;& z Helvetica;Wingdings?5 z Courier New"qhLELfj"lf:$24I I T 3qH(?NyY(Preparation of GST-RBD (for RhoA assays)Keith Burridge Marielle Yohe`                Oh+'0  8D ` l x )Preparation of GST-RBD (for RhoA assays) WorepKeith BurridgeGeiteitNormaluMarielle YoheG7riMicrosoft Word 10.0@=@뚛@P @&y՜.+,0, hp  University of North CarolinaRI : )Preparation of GST-RBD (for RhoA assays) Title  !"#$%&')*+,-./1234567:Root Entry FP<1Table&WordDocument&SummaryInformation((DocumentSummaryInformation80CompObjj  FMicrosoft Word Document MSWordDocWord.Document.89q