Immunoprecipitation of β-catenin from HeLa cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control (Sepharose® Bead Conjugate) #3423 (lane 3) and β-Catenin (D10A8) XP® Rabbit mAb (Sepharose® Bead Conjugate) (lane 2). Lane 1 is 10% input. Western blot analysis was performed using β-Catenin (L87A12) Mouse mAb #2698.
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This Cell Signaling Technology antibody is immobilized via covalent binding of primary amino groups to N-hydroxysuccinimide (NHS)-activated Sepharose® beads. β-Catenin (D10A8) XP® Rabbit mAb (Sepharose® Bead Conjugate) is useful for the immunoprecipitation of β-catenin. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated β-Catenin (D10A8) XP® Rabbit mAb #8480.
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
β-Catenin (D10A8) XP® Rabbit mAb (Sepharose® Bead Conjugate) recognizes endogenous levels of total β-catenin protein.
Human, Mouse, Rat, Monkey
Zebrafish, Bovine, Pig, Guinea Pig, Horse
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro714 of human β-catenin protein
β-Catenin is a key downstream effector in the Wnt signaling pathway (1). It is implicated in two major biological processes in vertebrates: early embryonic development (2) and tumorigenesis (3). CK1 phosphorylates β-catenin at Ser45. This phosphorylation event primes β-catenin for subsequent phosphorylation by GSK-3β (4-6). GSK-3β destabilizes β-catenin by phosphorylating it at Ser33, Ser37, and Thr41 (7). Mutations at these sites result in the stabilization of β-catenin protein levels and have been found in many tumor cell lines (8).
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