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Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP® Rabbit mAb (Alexa Fluor® 555 Conjugate) #9836
Gallery: Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP® Rabbit mAb (Alexa Fluor® 555 Conjugate) #9836
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
- Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
- Blocking Buffer (1X PBS / 5% normal goat serum (#5425) / 0.3% Triton™ X-100): To prepare 10 ml: add 0.5 ml normal goat serum and 0.5 ml 20X PBS to 9.0 ml dH2O, mix. While stirring, add 30 µl Triton™ X-100.
- Antibody Dilution Buffer: (1X PBS / 1% BSA / 0.3% Triton™ X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (#9998), mix.
- Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
B. Specimen Preparation - Cultured Cell Lines (IF-IC)
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.
- Allow cells to fix for 15 min at room temperature.
- Aspirate fixative, rinse three times in 1X PBS for 5 min each.
- Proceed with Immunostaining (Section C).
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
- Block specimen in Blocking Buffer for 60 min.
- While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
- Aspirate blocking solution, apply diluted primary antibody.
- Incubate overnight at 4°C.
- Rinse three times in 1X PBS for 5 min each.
- Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
- For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.
posted November 2006
revised November 2013
protocol id: 182
Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP® Rabbit mAb (Alexa Fluor® 555 Conjugate) detects endogenous levels of p38 MAPK only when phosphorylated at Thr180 and Tyr182. This antibody does not cross-react with the phosphorylated forms of either p44/42 MAPK or SAPK/JNK.Species Reactivity: Human, Mouse, Rat, Monkey, Mink, Pig, S. cerevisiae Species predicted to react based on 100% sequence homology: Hamster, Chicken, Zebrafish, Bovine, Pig
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr180/Tyr182 of human p38 MAPK protein.
This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 555 fluorescent dye and tested in-house for immunofluorescent analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP® Rabbit mAb #4511.
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).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc. XP® is a trademark of Cell Signaling Technology, Inc. The Alexa Fluor® dye antibody conjugates in this product are sold under license from Life Technologies Corporation for research use only, except for use in combination with DNA microarrays. The Alexa Fluor® dyes (except for Alexa Fluor® 430 dye) are covered by pending and issued patents. Alexa Fluor® is a registered trademark of Molecular Probes, Inc.