p38 MAPK activity of UV-treated or untreated C6 cell extracts was analyzed by IP/kinase assay. Cell extracts were immunoprecipitated with Phospho-p38 MAPK (Thr180/Tyr182) Mouse mAb (Sepharose® Bead Conjugate). In vitro kinase assays were performed using ATF-2 Fusion Protein #9224 as a substrate. Phosphorylation of ATF-2 at Thr71 was measured by Western blot using Phospho-ATF-2 (Thr71) Antibody.
|REACTIVITY||H M R Mk Sc|
20 mM Tris-HCl (pH 7.4)
150 mM NaCl
1 mM Na2EDTA
1 mM EGTA
1% Triton X-100
2.5 mM sodium pyrophosphate
1 mM β-glycerophosphate
1 mM Na3VO4
1 µg/ml leupeptin
Note: Please aliquot prior to first use.
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
Phospho-p38 MAPK (Thr180/Tyr182) Mouse mAb (Sepharose® Bead Conjugate) binds only p38 MAPK only when dually phosphorylated at Thr180 and Tyr182. This antibody was immobilized via conjugation of carbohydrates to cross-linked agarose hydrazide beads. This antibody does not significantly cross-react with phosphorylated SAPK/JNK or p44/42 MAPK, or with nonphosphorylated p38 MAPK.Species Reactivity:
Human, Mouse, Rat, Monkey, S. cerevisiaeSpecies predicted to react based on 100% sequence homology:
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues around Thr180/Tyr182 of human p38 MAP kinase.
p38 MAP kinase (MAPK), also called RK (1) or CSBP (2), is the mammalian orthologue of the yeast HOG kinase that participates in a signaling cascade controlling cellular responses to cytokines and stress (1-4). Four isoforms of p38 MAPK, p38α, β, γ (also known as Erk6 or SAPK3), and δ (also known as SAPK4) have been identified. Similar to the SAPK/JNK pathway, p38 MAPK is activated by a variety of cellular stresses including osmotic shock, inflammatory cytokines, lipopolysaccharide (LPS), UV light, and growth factors (1-5). MKK3, MKK6, and SEK activate p38 MAPK by phosphorylation at Thr180 and Tyr182. Activated p38 MAPK has been shown to phosphorylate and activate MAPKAP kinase 2 (3) and to phosphorylate the transcription factors ATF-2 (5), Max (6), and MEF2 (5-8). SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-imidazole) is a selective inhibitor of p38 MAPK. This compound inhibits the activation of MAPKAPK-2 by p38 MAPK and subsequent phosphorylation of HSP27 (9). SB203580 inhibits p38 MAPK catalytic activity by binding to the ATP-binding pocket, but does not inhibit phosphorylation of p38 MAPK by upstream kinases (10).
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