|H M R Hm Mk Mi Dm Z B Dg Pg Ce||Endogenous||42, 44||Rabbit IgG|
Immunoprecipitation of 3T3 cell lysates using p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb (Sepharose® Bead Conjugate) and Rabbit (DA1E) mAb IgG XP® Isotype Control (Sepharose® Bead Conjugate) #3423. The western blot was probed using p44/42 MAPK (Erk1/2) (3A7) Mouse mAb #9107.Learn more about how we get our images
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
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.
p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb (Sepharose® Bead Conjugate) detects endogenous levels of total p44/42 MAPK (Erk1/2) protein. The antibody does not cross-react with JNK/SAPK or p38 MAP kinase. The addition of 0.1% SDS to the immunoprecipitation reaction may improve pulldown efficiency. Under some conditions this conjugate may immunoprecipitate p44/Erk1 preferentially over p42/Erk2.
Human, Mouse, Rat, Hamster, Monkey, Mink, D. melanogaster, Zebrafish, Bovine, Dog, Pig, C. elegans
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the C-terminus of rat p44 MAP kinase.
This Cell Signaling Technology antibody is immobilized via covalent binding of primary amino groups to N-hydroxysuccinimide (NHS)-activated Sepharose® beads. p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb (Sepharose® Bead Conjugate) is useful for immunoprecipitation assays. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb #4695.
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.
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.
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