Peptide Competition and Upregulation Extracts of NIH3T3 cells untreated (1) or treated with 50ng/mL PDGF for 15 minutes (2-5) were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 4% BSA-TBST buffer for one hour at room temperature, then incubated with the MEK1 [pT386] antibody in a 1% BSA-TBST buffer for two hours at room temperature, following prior incubation with: no peptide (1, 2), the non-phosphopeptide corresponding to the phosphopeptide immunogen (3), a generic phosphothreonine-containing peptide (4), or the phosphopeptide immunogen (5). After washing, the membrane was incubated with goat F(ab and quote;)2 anti-rabbit IgG HRP conjugate (Cat. no. ALI4404) and signals were detected using the Pierce SuperSignal™ method.The data show that only the phosphopeptide corresponding to MEK1 [pT386] blocks the antibody signal, demonstrating the specificity of the antibody. The data also show the upregulation of MEK1 [pT386] upon PDGF treatment in this cell system.
|Tested species reactivity||Human, Mouse, Rat|
|Host / Isotype||Rabbit / IgG|
|Immunogen||The antiserum was produced against a chemically synthesized phosphopeptide derived from a region of human MEK1 that contains threonine 386. The sequence is conserved in many species including mouse, rat, chimp, hamster, and rabbit.|
|Purification||Antigen affinity chromatography|
|Storage buffer||Dulbecco's PBS, pH 7.3, with 50% glycerol, 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Western Blot (WB)||Assay Dependent|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
MAP2K1 is a dual specificity mitogen-activated protein kinase (MAPK) kinase. It is an essential component of the MAP kinase pathway and stimulates the enzymatic activity of MAP kinases in response to various signals. Growth factors, cytokines, and proto-oncogenes transduce their signals through the activation of RAS, which leads to activation of the serine/threonine kinase RAF, and then to activation of MAPK kinase. MAP2K1 is activated by the Raf family member p74raf-1, through phosphorylation of serine residues at positions 217 and 221 in the activation loop.
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