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Phospho-p38 MAPK (Thr180/Tyr182) (28B10) Mouse mAb (Alexa Fluor® 647 Conjugate) #4552
Flow cytometric analysis of THP-1 cells, untreated (blue) or treated with Anisomycin (25ug/ml, 30 min; green) using Phospho-p38 MAPK (Thr180/Tyr182) (28B10) Mouse mAb (Alexa Fluor® 647 Conjugate) (solid lines) or concentration-matched Mouse (MOPC-21) mAb IgG1 Isotype Control (Alexa Fluor® 647 Conjugate) #4843 (dashed lines).Learn more about how we get our images
Gallery: Phospho-p38 MAPK (Thr180/Tyr182) (28B10) Mouse mAb (Alexa Fluor® 647 Conjugate) #4552
Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
- 16% Formaldehyde (methanol free).
- 100% methanol.
- Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
- Collect cells by centrifugation and aspirate supernatant.
- Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
- Fix for 15 min at room temperature.
- Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS.
- Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
- Incubate 30 min on ice.
- Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.
- Aliquot desired number of cells into tubes or wells.
- Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
- Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
- Incubate for 1 hr at room temperature. Protect from light.
- Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
- Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).
E. Optional DNA Dye
- Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
- Incubate for at least 5 min at room temperature.
- Analyze cells in DNA staining solution on flow cytometer.
posted July 2009
revised June 2017
Phospho-p38 MAPK (Thr180/Tyr182) (28B10) Mouse mAb (Alexa Fluor® 647 Conjugate) detects endogenous levels of p38 MAP kinase only when dually phosphorylated at Thr180 and Tyr182. This antibody does not appreciably cross-react with the corresponding phosphorylated forms of either p44/42 MAPK (Erk1/2) or SAPK/JNK.Species Reactivity: Human, Mouse, Rat, Monkey, S. cerevisiae Species predicted to react based on 100% sequence homology: Zebrafish
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr180/Tyr182 of human p38 MAP kinase. This antibody was conjugated to Alexa Fluor® 647 under optimal conditions with an F/P ratio of 2-6. The Alexa Fluor® 647 dye is maximally excited by red light (e.g. 633 nm He-Ne laser). Antibody conjugates of the Alexa Fluor® 647 dye produce bright far-red-fluorescence emission, with a peak at 665 nm.
This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 647 fluorescent dye and tested in-house for direct flow cytometric analysis of human and mouse cells. The unconjugated antibody #9216 reacts with human, mouse, rat, D. melanogaster, monkey, S.cerevisiae and zebra fish phospho-p38 MAPK (Thr180/Tyr182). CST expects that Phospho-p38 MAPK (Thr180/Tyr182) (28B10) Mouse mAb (Alexa Fluor® 647 Conjugate) will also recognize phospho-p38 MAPK (Thr180/Tyr182) in these species.
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. 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.