Immunoprecipitation of Jurkat cell lysates, untreated or treated with Calyculin A, using Phospho-Akt Substrate (RXXS*/T*) (110B7E) Rabbit mAb (Magnetic Bead Conjugate) (lane 1 and 2) and Rabbit (DA1E) mAb IgG XP® Isotype Control (Magnetic Bead Conjugate) #8726 (lane 3 and 4). The western blot was probed using Phospho-Akt Substrate (RXXS*/T*) (110B7E) Rabbit mAb (HRP Conjugate) #6950.
This Cell Signaling Technology antibody is immobilized by the covalent reaction of formylbenzamide-modified antibody with hydrazide-activated magnetic bead.
Phospho-Akt Substrate (RXXS*/T*) (110B7E) Rabbit mAb (Magnetic Bead Conjugate) is useful for immunoprecipitation of phosphorylated Akt substrate proteins.
Supplied in PBS Buffer (pH 7.2), 0.1% Tween® 20. Store at 4°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: 408
Phospho-Akt Substrate (RXXS*/T*) (110B7E) Rabbit mAb (Magnetic Bead Conjugate) recognizes peptides and proteins containing phospho-Ser/Thr preceded by Arg at the -3 position. There is some preference observed for peptides that contain phospho-Ser/Thr preceded by Arg 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.)
All Species Expected
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.