Immunoprecipitation of HeLa cell lysates using Rabbit (DA1E) mAb IgG XP® Isotype Control (Sepharose Bead Conjugate) #3423 (lane 1) and Rictor (D16H9) Rabbit mAb (Sepharose Bead Conjugate) (lane 2). The western blot was probed using Rictor (D61H9) Rabbit mAb #9476.
This Cell Signaling Technology antibody is immobilized via covalent binding of primary amino groups to N-hydroxysuccinimide (NHS)-activated sepharose beads. Rictor (D16H9) Rabbit mAb (Sepharose Bead Conjugate) is useful for immunoprecipitation assays. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Rictor (D16H9) Rabbit mAb #9476.
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
Rictor (D16H9) Rabbit mAb (Sepharose Bead Conjugate) detects endogenous levels of total rictor protein.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the sequence around Leu1121 of human rictor protein.
Cell growth is a fundamental biological process whereby cells accumulate mass and increase in size. The mammalian TOR (mTOR) pathway regulates growth by coordinating energy and nutrient signals with growth factor-derived signals (1). mTOR is a large protein kinase with two different complexes. One complex contains mTOR, GβL and raptor, which is a target of rapamycin. The other complex, insensitive to rapamycin, includes mTOR, GβL, Sin1, and rictor (1). The mTOR-rictor complex phosphorylates Ser473 of Akt/PKB in vitro (2). This phosphorylation is essential for full Akt/PKB activation. Furthermore, an siRNA knockdown of rictor inhibits Ser473 phosphorylation in 3T3-L1 adipocytes (3). This complex has also been shown to phosphorylate the rapamycin-resistant mutants of S6K1, another effector of mTOR (4).
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