REACTIVITY | SENSITIVITY | MW (kDa) | SOURCE |
---|---|---|---|
H M R Mk Dm Pg Sc | Endogenous | 43 | Rabbit |
Western blot analysis of extracts from C6 cells, untreated or anisomycin-treated, and NIH/3T3 cells, untreated or UV-treated, using Phospho-p38 MAPK (Thr180/Tyr182) Antibody (upper) or p38 MAPK Antibody #9212 (lower).
Learn more about how we get our imagesWestern blot analysis of extracts from UV-treated NIH/3T3 cells, using Phospho-p38 MAPK (Thr180/Tyr182) Antibody (upper) or control p38 MAPK Antibody #9212 (lower).
Learn more about how we get our imagesConfocal immunofluorescent analysis of HeLa cells -/+ UV light, labeled with Phospho-p38 MAPK (green). Absence of staining in untreated cells (left) and nuclear localization in treated cells (right). Red = Actin filaments (phalloidin).
Learn more about how we get our imagesFlow cytometric analysis of Jurkat cells, untreated (green) or anisomycin treated (blue), using Phospho-p38 MAPK (Thr180/Tyr182) Antibody compared to a nonspecific negative control antibody (red).
Learn more about how we get our imagesFor 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.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
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: Loading of prestained molecular weight markers (#13953, 5 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* 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 October 2016
Protocol Id: 10
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 equivalent grade water.
10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH2O, mix.
NOTE: Add 1 mM PMSF (#8553) immediately prior to 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 2008
revised November 2013
Protocol Id: 409
Achieve higher quality immunofluorescent images using the efficient and cost-effective, pre-made reagents in our #12727 Immunofluorescence Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:
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.
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.
posted November 2006
revised November 2013
Protocol Id: 24
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
NOTE: Count cells using a hemocytometer or alternative method.
posted July 2009
revised June 2017
Protocol Id: 404
Application | Dilutions |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:50 |
Immunofluorescence (Immunocytochemistry) | 1:800 |
Flow Cytometry | 1:400 |
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
Phospho-p38 MAPK (Thr180/Tyr182) Antibody detects endogenous levels of p38 MAPK only when activated by phosphorylation at threonine 180 and tyrosine 182. This antibody does not cross-react with the phosphorylated forms of either p42/44 MAPK or SAPK/JNK. It will also react with p38 singly phosphorylated at Thr180 and singly phosphorylated at Tyr182.
Human, Mouse, Rat, Monkey, D. melanogaster, Pig, S. cerevisiae
Hamster, Zebrafish, Bovine
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues around Thr180/Tyr182 of human p38 MAPK. Antibodies are purified by protein A and peptide affinity chromatography.
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).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Explore pathways related to this product.
Product # | Size | Price |
---|---|---|
9211S | 200 µl (20 western blots) | $ 297.0 |
9211L | 600 µl (60 western blots) | $ 702.0 |