аЯрЁБс>ўџ 79ўџџџ6џџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџьЅС@ №ПЦ$bjbjюFюF &Œ,Œ,c џџџџџџˆ:::::::N–––– ЂNаЄЪЪЪЪЪЪЪЪ!!!!!!$t RЦ"АEE:ЪЪЪЪЪE::ЪЪŠКККЪ:Ъ:ЪКЪККNяЈ::їЪО №iН oŒХ–мR—0 0аЧ0v#.‚v#`їNN::::v#:ї(ЪЪКЪЪЪЪЪEENNЄђЄА NNђPreparation of GST-PBD beads (for Cdc42 and Rac assays) 1) Growth of bacteria prepare ON culture of pGEX GST-PBD (50 mL LB with 100 Еg/mL ampicillin), incubate ON @ 37кC dilute ON culture 1:200 into a final volume of 1 L LB with 100 Еg/mL ampicillin grow culture for a further 1-2 hours @ 37кC (OD @ 600 nm should be about 0.6-0.8) induce protein expression by adding 1 mM IPTG for 5 hours @ 37кC 2) Purification of GST-PBD on Glutathione Sepharose 4B pellet bacteria by centrifugation @ 4000 rpm for 10 minutes @ 4кC resuspend pellet in lysis buffer: 20 mM HEPES, pH 7.5 120 mM NaCl 10% glycerol 2 mM EDTA 1 mM PMSF 10 ug/mL each of aprotinin and leupeptin Lyse sample with emulsiflex spin @ 15000 rpm @ 4кC for 30 minutes to supernatant add NP-40 to 0.5% add 600 ЕL of a 50% slurry of glutathione-Sepharose 4B beads incubate 1 hour @ 4кC with rotation wash beads 5X in lysis buffer with 0.5% NP-40 wash beads 3X in lysis buffer with no NP-40 store @ 4кC for up to 1 week as a 50% slurry in lysis buffer with no NP-40 3) analysis of GST-PBD product measure the protein concentration of the beads (should be 10 mg/mL) run fusion protein on SDS-PAGE gel and stain with commassie (fusion protein should run around 33 kDa) Rac1 and Cdc42 Activity Assays use enough cells to yield 800-1000 Еg total protein (one 10 cm2 plate is enough for most cell types) lyse cells in 300 ЕL of Buffer B 50 mM Tris, pH 7.6 150 mM NaCl 1% Triton X-100 20 mM MgCl2 + inhibitors (1 mM PMSF, 10 Еg/ml leupeptin, 10 Еg/ml aprotinin) sonicate lysates for 10 second lysates become viscous spin for 10 minutes @ 14,000 rpm @ 4кC measure protein concentration and dilute with lysis buffer equal concentration and volume in all the samples. Take off 10% of the concentration-equalized lysates to arШ V Ь Ј : p dhњўBaаё/0nvЋЏЗИdzЦШN\№Ц$ѕэхэхэхэхэмэмэхэѕэгэмэхэЪэхэхэШэхэмэUhSarH*OJQJhSarH*OJQJhSar>*OJQJh@ZOJQJhSarOJQJhSar5>*OJQJ$pržV і š  Š  R z – Д Ь ф : r њѕѓхзЩЛѓ­­Ѓѓѓѓѓѓ• & F Цhа„а^„а „а„а^„а`„а & F Цhа„а^„а & F Цh8„8^„8 & F Цh8„8^„8 & F Цh8„8^„8 & F Цh8„8^„8$a$gd†?hЦ$ўr О zТv%iЯб№ёVw‹™ЋЙќёёёёёёёяссигиХХПяяяя„а`„а & F Цhа„а^„аgd@Z$„а`„аa$ & F Цhа„а^„а & F Цhа„а^„аќdВNvЮHа6 ‚ †!ˆ!Ш"<$>$Ц$ёёёёёёёёёёёяяяяя & F Цhа„а^„а new tube for lysate loading controls. add 120 Еg GST-PBD* incubate @ 4кC with rotation for 30 minutes wash beads 3X in Buffer B carefully aspirate off supernatant suspend beads in 30-90 ЕL of SDS-PAGE sample buffer, boil 5 minutes run sample on 15% SDS-PAGE gel, transfer to PVDF** block 5-15 minutes in TBS-T + 5% milk blot GST-PBD pull-downs with mAb anti-Rac (1:1000) or mAb anti-Cdc42 (1:250). All antibodies are from Transduction Laboratories. *Rac1 and Cdc42 activity can be assessed from the same GST-PBD pull-down. To do this use 1/4 of the sample for the Rac1 blot and 3/4 of sample for Cdc42 blot. ** 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 1-3% of lysate for Rac, 3-10% for Cdc42. 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