Immunoprecipitation of NIH/3T3 cell lysates, untreated or PDGF-treated, using Phospho-Akt Substrate (RXXS*/T*) (110B7E) Rabbit mAb (Sepharose® Bead Conjugate) compared to supernatants collected after immunoprecipitation from the same lysate. The western blot was probed using Phospho-Akt Substrate (RXXS*/T*) (110B7E) Rabbit mAb #9614.
This Cell Signaling Technology antibody is immobilized via covalent binding of primary amino groups to N-hydroxysuccinimide (NHS)-activated Sepharose® beads. Phospho-Akt Substrate (RXXS*/T*) (110B7E) Rabbit mAb (Sepharose® Bead Conjugate) is useful for the immunoprecipitation of phosphorylated Akt substrate proteins.
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol. Store at –20°C. Do not aliquot the antibodies.
This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
10X Cell Lysis Buffer: (#9803) 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml Leupeptin
NOTE: CST recommends adding 1 mM PMSF (#8553) before use*.
Proceed to one of the following specific set of steps.
NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (L57A3) mAb (#3677) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).
posted December 2007
Protocol Id: 27
Phospho-(Ser/Thr) Akt Substrate Motif (RXXS*/T*) (110B7) Rabbit mAb (Sepharose® Bead Conjugate) recognizes peptides and proteins containing phospho-serine/threonine preceded by arginine at the -3 position. There is some preference observed for peptides that contain phospho-serine/threonine preceded by arginine at both positions -5 and -3. (U.S. Patent No's.: 6,441,140; 6,982,318; 7,259,022; 7,344,714; U.S.S.N. 11,484,485; and all foreign equivalents.)
Monoclonal antibody is produced by immunizing animals with synthetic phospho-Akt substrate peptides.
An important class of kinases, referred to as Arg-directed kinases or AGC-family kinases, includes cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), protein kinase C, Akt, and RSK. These kinases share a substrate specificity characterized by Arg at position -3 relative to the phosphorylated Ser or Thr (1,2). Akt plays a central role in mediating critical cellular responses including cell growth and survival, angiogenesis, and transcriptional regulation (3-5). While a number of Akt substrates are known (such as GSK-3, Bad, and caspase-9) many important substrates await discovery. Akt phosphorylates substrates only at Ser/Thr in a conserved motif characterized by Arg at positions -5 and -3 (6). Phospho-Akt substrate-specific antibodies from Cell Signaling Technology are powerful tools for investigating the regulation of phosphorylation by Akt and other Arg-directed kinases, as well as for high throughput kinase drug discovery.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
Use of Cell Signaling Technology (CST) Motif Antibodies within certain methods (e.g., U.S. Patents No. 7,198,896 and 7,300,753) may require a license from CST. For information regarding academic licensing terms please have your technology transfer office contact CST Legal Department at CST_ip@cellsignal.com. For information regarding commercial licensing terms please contact CST Pharma Services Department at email@example.com.