Science Catalog 2012 Life Cell Signaling A. 1 2 B. 1 2 kDa 250 – 98 – 64 – 50 – – Phospho-Akt – Akt 36 – 30 – 16 – 6 – 4 – 1 2 3 4 5 6 7 8 9 10 11 Detection of phosphorylated Akt by Western blot analysis with Anti-pS473 Akt pAb. Panel A. NIH/3T3 total cell extract (10μg per lane) was resolved by polyacrylamide gel electrophoresis and blotted onto nitrocellulose. Lane 1, untreated cells; lane 2, cells pretreated with PDGF (Invitrogen) at 50ng/ml for 20 minutes. Anti-pS473 Akt pAb (Cat.# G7441) was used at a 1:2,500 dilution. The blot was probed with Donkey Anti-Rabbit IgG (H+L), HRP, Anti-ACTIVE® Qualified pAb (Cat.# V7951) at 1:10,000 dilution followed by chemiluminescent detection. Panel B. A pan-Akt pAb (New England Biolabs) reveals total Akt in both stimulated and unstimulated NIH/3T3 cell extracts. Secondary antibody and detection methods were the same as those used in Panel A. Anti-ACTIVE® CaM KII pAb, Rabbit, (pT286) Product Anti-ACTIVE® CaM KII pAb, Rabbit, (pT286) For Research Use Only. Not for Use in Diagnostic Procedures. Description: This polyclonal antibody (pAb) is specific for the multifunctional calcium/calmodulin-dependent protein kinase CaM kinase II (CaM KII) that is phosphorylated on threonine 286 (pT286). The Anti-ACTIVE® CaM KII pAb was raised against the phosphothreonine-containing peptide. Features: • Specificity: Preferentially detects CaM KII autophosphorylated on threonine 286. • Immunogen: Threonine-phosphorylated peptide corresponding to the phosphorylated Thr286 region of the mammalian calcium/calmodulindependent protein kinase. • Antibody Form: Affinity-purified rabbit IgG; supplied in 10mM sodium phosphate (pH 7.4), 20mM NaCl. • Value: When used at the recommended dilution of 1:5,000, it generates up to 200ml of blotting solution sufficient for 20 Western blots of 10ml each. Storage Conditions: Store at –20°C. Protocol Anti-ACTIVE® CaM KII pAb, (pT286) and Anti-ACTIVE® Qualified Secondary Antibody Conjugates Technical Bulletin Part# TB264 Size Cat.# Price (Fr) 40 µl V1111 848.00 Detection of CaM KII by Anti-ACTIVE® CaM KII pAb in Western analysis of rat brain homogenate. Lanes 2, 4, 6, 8 and 10 contain autophosphorylated (+) brain cytosolic protein in the amounts shown; lanes 3, 5, 7, 9 and 11 contain nonphosphorylated (–) brain cytosolic protein in the amounts shown. The presence of the autophosphorylated CaM KII was detected using the Anti-ACTIVE® CaM KII pAb diluted 1:5,000. Panel A Panel B Panel C + – + – + – + – + – 0.5µg 2.5µg 5µg 12.5µg 25µg – Phospho T286 CaM KII Immunocytochemical detection of autophosphorylated CaM KII in PC12 cells with Anti-ACTIVE® CaM KII pAb. PC12 cells were adhered to slides coated with collagen, fixed in 10% paraformaldehyde for 30 minutes, rinsed in PBS and permeabilized in methanol for 10 minutes at –20˚C. The cells were then blocked in 1% BSA in PBS for 45 minutes followed by 2% horse serum in PBS for 60 minutes. Cells were incubated overnight at 4˚C with (Panel A) Ab alone, (Panel B) Ab preincubated with a nonphosphorylated CaM KII peptide fragment (1μg/ml) or (Panel C) Ab preincubated with a phosphorylated CaM KII peptide fragment (1μg/ml). The Anti-ACTIVE® CaM KII pAb was used at 1:500 dilution and preincubated with peptide for 8 hours at 4˚C. After incubation with the Anti-ACTIVE® CaM KII pAb or Ab/peptide mixture, the cells were rinsed in PBS and incubated with a donkey anti-rabbit FITC-conjugated secondary Ab (1:500) for 60 minutes at room temperature. Staining was visualized with a Zeiss® fluorescence microscope. The results demonstrate that preincubation of the Anti-ACTIVE® CaM KII pAb with phosphorylated CaM KII peptide completely abolishes immunostaining (Panel C), but preincubation with nonphosphorylated CaM KII peptide has no effect on immunostaining (Panel A versus Panel B). Protocols developed and performed at Promega. 166 For complete and up-to-date product information visit: www.promega.com/catalog 2810TA10_9A 2055TA02_8A 2057TA02_8A Pagina 169
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