REACTIVITY | SENSITIVITY | MW (kDa) | SOURCE |
---|---|---|---|
H M R Hm Mk Mi Dm Z B Pg Ce | Endogenous | 42, 44 | Rabbit |
Specificity and sensitivity of Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Antibody. The antibody reacts specifically with as little as 0.25 ng of phosphorylated p42 MAP kinase and does not cross-react with up to 4 µg of nonphosphorylated p42 MAP kinase.
Learn more about how we get our imagesWestern blot analysis of whole-cell extracts from unstarved wild-type mouse embryonic fibroblasts (MEFs) treated with the indicated combinations of basic Fibroblast Growth Factor (bFGF #9952, 100 ng/ml for 30 minutes), Platelet-Derived Growth Factor (PDGF #9909, 100 ng/ml for 30 minutes), MEK1 Inhibitor (PD98059 #9900, 50 µM, 2 hour pre-treatment), and MEK1/2 Inhibitor (U0126 #9903, 10 µM, 2 hour pre-treatment), using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Antibody #9101 (upper panel) and p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb #4695 (lower panel).
Learn more about how we get our imagesConfocal immunofluorescent analysis of NIH/3T3 cells either U0126-treated (left) or PDGF-treated (right) and labeled with Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Antibody (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin #8953 (red).
Learn more about how we get our imagesFlow cytometric analysis of Jurkat cells, untreated (green) or PMA-treated (blue), using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Antibody compared to a nonspecific negative control antibody (red).
Learn more about how we get our imagesPhosphorylated MEK and Erk were assayed in human peripheral blood lymphocytes stimulated with PMA in the presence or absence of the Raf inhibitor BAY 37-9751 or the MEK inhibitor U0126 #9903. BAY 37-951 blocked PMA-stimulated phosphorylation of both MEK and Erk, consistent with inhibition at the level of Raf, while U0126 blocked phosphorylation of Erk only, consistent with inhibition at the level of MEK. From Chow, S. et al. (2001) Cytometry 46, 72-78.
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 utilizing magnetic separation.
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.
A cell lysate pre-clearing step is highly recommended to reduce non-specific protein binding to the Protein A Magnetic beads. Pre-clear enough lysate for test samples and isotype controls.
IMPORTANT: Pre-wash #73778 magnetic beads just prior to use:
Carefully remove the buffer once the solution is clear. Add 500 μl of 1X cell lysis buffer to the magnetic bead pellet, briefly vortex to wash the beads. Place tube back in magnetic separation rack. Remove buffer once solution is clear. Repeat washing step once more.
IMPORTANT: The optimal lysate concentration will depend on the expression level of the protein of interest. A starting concentration between 250 μg/ml-1.0 mg/ml is recommended.
IMPORTANT: Appropriate isotype controls are highly recommended in order to show specific binding in your primary antibody immunoprecipitation. Use Normal Rabbit IgG #2729 for rabbit polyclonal primary antibodies, Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 for rabbit monoclonal primary antibodies, and Mouse (G3A1) mAb IgG1 Isotype Control #5415 for mouse monoclonal primary antibodies. Isotype controls should be concentration matched and run alongside the primary antibody samples
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) (D4W3E) mAb (#45262) 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 October 2017
Protocol Id: 410
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:
NOTE: Cells should be grown, treated, fixed and stained directly in multiwell plates, chamber slides or on coverslips.
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 December 2010
Protocol Id: 32
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) | 1:250 |
Flow Cytometry | 1:200 |
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-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Antibody detects endogenous levels of p44 and p42 MAP Kinase (Erk1 and Erk2) when phosphorylated either individually or dually at Thr202 and Tyr204 of Erk1 (Thr185 and Tyr187 of Erk2). The antibody does not cross-react with the corresponding phosphorylated residues of either JNK/SAPK or p38 MAP Kinase, and does not cross-react with non-phosphorylated Erk1/2.
Human, Mouse, Rat, Hamster, Monkey, Mink, D. melanogaster, Zebrafish, Bovine, Pig, C. elegans
Chicken
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr202/Tyr204 of human p44 MAP kinase.
Mitogen-activated protein kinases (MAPKs) are a widely conserved family of serine/threonine protein kinases involved in many cellular programs, such as cell proliferation, differentiation, motility, and death. The p44/42 MAPK (Erk1/2) signaling pathway can be activated in response to a diverse range of extracellular stimuli including mitogens, growth factors, and cytokines (1-3), and research investigators consider it an important target in the diagnosis and treatment of cancer (4). Upon stimulation, a sequential three-part protein kinase cascade is initiated, consisting of a MAP kinase kinase kinase (MAPKKK or MAP3K), a MAP kinase kinase (MAPKK or MAP2K), and a MAP kinase (MAPK). Multiple p44/42 MAP3Ks have been identified, including members of the Raf family, as well as Mos and Tpl2/COT. MEK1 and MEK2 are the primary MAPKKs in this pathway (5,6). MEK1 and MEK2 activate p44 and p42 through phosphorylation of activation loop residues Thr202/Tyr204 and Thr185/Tyr187, respectively. Several downstream targets of p44/42 have been identified, including p90RSK (7) and the transcription factor Elk-1 (8,9). p44/42 are negatively regulated by a family of dual-specificity (Thr/Tyr) MAPK phosphatases, known as DUSPs or MKPs (10), along with MEK inhibitors, such as U0126 and PD98059.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Alexa Fluor is a registered trademark of Life Technologies Corporation.
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Product # | Size | Price |
---|---|---|
9101S | 200 µl (20 western blots) | $ 297.0 |
9101L | 600 µl (60 western blots) | $ 702.0 |