Western blot analysis of p38 MAPK Control Cell Extracts #9213, untreated or anisomycin-treated, using Phospho-p38 MAPK (Thr180/Tyr182) (3D7) Rabbit mAb (Biotinylated).
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This Cell Signaling Technology (CST) antibody is conjugated to biotin under optimal conditions. The unconjugated Phospho-p38 MAPK (Thr180/Tyr182) (3D7) Rabbit mAb #9215 reacts with human, mouse, rat, monkey, pig, S. cerevisiae, and D. melanogaster phospho-p38 MAP kinase (Thr180/Tyr182). CST expects that Phospho-p38 MAP Kinase (Thr180/Tyr182) (3D7) Rabbit mAb (Biotinylated) will also recognize phospho-p38 MAP kinase (Thr180/Tyr182) in these species.
Supplied in 136 mM NaCl, 2.6 mM KCI, 12 mM sodium phosphate (pH 7.4) dibasic, 2 mg/ml BSA, and 50% glycerol. Store at –20°C. Do not aliquot the antibodies.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
Do not add Anti-biotin, HRP-linked Antibody for detection of biotinylated protein markers. There is no need. The Streptavidin-HRP will also visualize the biotinylated markers.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised June 2016
Protocol Id: 266
Phospho-p38 MAP Kinase (Thr180/Tyr182) (3D7) Rabbit mAb (Biotinylated) detects endogenous levels of p38 MAPK only when phosphorylated at both Thr180 and Tyr182. This antibody does not cross-react with the phosphorylated forms of either p42/44 MAPK or SAPK/JNK.Species Reactivity:
Human, Mouse, Rat, Monkey, D. melanogaster, Pig, S. cerevisiaeSpecies predicted to react based on 100% sequence homology:
Hamster, Mink, Zebrafish, Bovine
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr180/Tyr182 of human p38 MAPK.
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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U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.