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Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP® Rabbit mAb (Alexa Fluor® 594 Conjugate) #8632
Gallery: Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP® Rabbit mAb (Alexa Fluor® 594 Conjugate) #8632
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified 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.
- Permeabilization Buffer (1X PBS/0.2% Triton X-100):
To prepare 25 ml, add 2.5 ml 10X PBS, and 22.5 ml dH2O and mix well. While stirring, add 50 µl Triton X-100.
- Image-iT™ FX Signal Enhancer (#11932)
- Blocking Buffer (1X PBS / 5% normal serum / 0.3% Triton™ X-100): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425)) and 0.5 mL 20X PBS to 9.0 mL dH2O, mix well. 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 in 1X PBS.
NOTE: Formaldehyde is toxic, use only in fume hood.
- Allow cells to fix for 15 minutes at room temperature.
- Aspirate fixative, rinse three times in 1X PBS for 5 minutes 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.
- Permeabilize the cells in Permeabilization Buffer for 5 minutes at room temperature.
- Rinse three times in 1X PBS for 5 minutes each.
- Apply 3–4 drops of Image-iT™ FX Signal Enhancer (#11932) and incubate for 30 minutes at room temperature.
- Rinse three times with 1X PBS for 5 minutes each.
- Block specimen in Blocking Buffer for 60 minutes.
- 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 minutes each.
- Coverslip slides with Prolong® Gold Antifade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
- For best results, examine specimens immediately using appropriate excitation wavelength. For long-term storage, store slides flat at 4°C protected from light.
posted Februay 2011
protocol id: 6
Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP® Rabbit mAb (Alexa Fluor® 594 Conjugate) recognizes 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® 594 fluorescent dye and tested in-house for immunofluorescent analysis in human cells. This 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 registered 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.