Immunoprecipitation of extracts from HeLa cells, treated with either U0126 #9903 (10uM, 1 hour, lanes 1, 3, and 5) or TPA #4147 (200uM 20 minutes, lanes 2, 4, and 6) using #3423 Rabbit (DA1E) mAb IgG XP® Isotype Control (Sepharose® Bead Conjugate) (lanes 1 and 2) or Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D6A9) Rabbit mAb (Sepharose® Bead Conjugate) (lanes 3 and 4). Lanes 5 and 6 are 10% input. Western blot analysis performed using #8544 Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb (HRP Conjugate).
|REACTIVITY||H M R Mk|
|MW (kDa)||42, 44|
This Cell Signaling Technology antibody is immobilized via covalent binding of primary amino groups to N-hydroxysuccinimide (NHS)-activated Sepharose® beads.
Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Antibody (A4M8T) Rabbit mAb (Sepharose® Bead Conjugate) is useful for the immunoprecipitation of phospho-p44/p42 MAPK. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Antibody #9101.
|MW (kDa)||42, 44|
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 antibody.
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-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (A4M8T) Rabbit mAb (Sepharose® Bead Conjugate) detects endogenous levels of p44 and p42 MAP Kinase (Erk1 and Erk2) when phosphorylated either individually or dually at Thr202 and Tyr204 of Erk1 (Thr185 and Tyr187 of Erk2). The antibody does not cross-react with the corresponding phosphorylated residues of either JNK/SAPK or p38 MAP Kinase, and does not cross-react with non-phosphorylated Erk1/2.
Human, Mouse, Rat, Monkey
Monoclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr202/Tyr204 of human p44 MAP kinase.
Mitogen-activated protein kinases (MAPKs) are a widely conserved family of serine/threonine protein kinases involved in many cellular programs, such as cell proliferation, differentiation, motility, and death. The p44/42 MAPK (Erk1/2) signaling pathway can be activated in response to a diverse range of extracellular stimuli including mitogens, growth factors, and cytokines (1-3), and research investigators consider it an important target in the diagnosis and treatment of cancer (4). Upon stimulation, a sequential three-part protein kinase cascade is initiated, consisting of a MAP kinase kinase kinase (MAPKKK or MAP3K), a MAP kinase kinase (MAPKK or MAP2K), and a MAP kinase (MAPK). Multiple p44/42 MAP3Ks have been identified, including members of the Raf family, as well as Mos and Tpl2/COT. MEK1 and MEK2 are the primary MAPKKs in this pathway (5,6). MEK1 and MEK2 activate p44 and p42 through phosphorylation of activation loop residues Thr202/Tyr204 and Thr185/Tyr187, respectively. Several downstream targets of p44/42 have been identified, including p90RSK (7) and the transcription factor Elk-1 (8,9). p44/42 are negatively regulated by a family of dual-specificity (Thr/Tyr) MAPK phosphatases, known as DUSPs or MKPs (10), along with MEK inhibitors, such as U0126 and PD98059.
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