The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated ERK5. The final product is generated by affinity chromatography using an ERK5-derived peptide that is phosphorylated at threonine 218 and tyrosine 220.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/1000. Predicted molecular weight: 88 kDa. Due to the low abundance and low levels of activation of endogenous ERK5, over-expression or immunoprecipitation may be required.
Use at an assay dependent concentration.
Plays a role in various cellular processes such as proliferation, differentiation and cell survival. The upstream activator of MAPK7 is the MAPK kinase MAP2K5. Upon activation, it translocates to the nucleus and phosphorylates various downstream targets including MEF2C. EGF activates MAPK7 through a Ras-independent and MAP2K5-dependent pathway. May have a role in muscle cell differentiation. May be important for endothelial function and maintenance of blood vessel integrity. MAP2K5 and MAPK7 interact specifically with one another and not with MEK1/ERK1 or MEK2/ERK2 pathways.
Expressed in many adult tissues. Abundant in heart, placenta, lung, kidney and skeletal muscle. Not detectable in liver.
Belongs to the protein kinase superfamily. CMGC Ser/Thr protein kinase family. MAP kinase subfamily. Contains 1 protein kinase domain.
The second proline-rich region may interact with actin targeting the kinase to a specific location in the cell. The TXY motif contains the threonine and tyrosine residues whose phosphorylation activates the MAP kinases.
Dually phosphorylated on Thr-219 and Tyr-221, which activates the enzyme (By similarity). Autophosphorylated in vitro on threonine and tyrosine residues when the C-terminal part of the kinase, which could have a regulatory role, is absent.
Cytoplasm. Nucleus. Translocates to the nucleus upon activation.
Western blot - ERK5 (phospho T218 + Y220) antibody (ab5686)
Predicted band size: 88 kDa
Extracts prepared from HEK293 cells transiently transfected with plasmids expressing ERK5 kinase domain (ERK5kin) and constitutively activated MEK5D-D were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 5% BSA TBST buffer overnight at 4oC, then were incubated with the ab5686 antibody for two hours at room temperature in a 3% BSATBST buffer, following prior incubation with: no peptide (1), the non-phosphopeptide corresponding to the immunogen (2), a generic phosphothreonine-containing peptide (3), a generic phosphotyrosine-containing peptide (4), the phosphopeptide derived from the corresponding region of ERK1&2 (5), or, the phosphopeptide immunogen (6). After washing, membranes were incubated with goat F(ab’ 2 anti-rabbit IgG alkaline phosphatase conjugate and bands were detected using the Tropix WesternStarTM detection method. The data show that while there is some cross-reactivity with ERK1&2, only the phosphopeptide corresponding to ERK5 [pTpY218/220] completely blocks the antibody signal, demonstrating the specificity of the antibody. NOTE: The antibody signal appears at ~50 kDa as this is the molecular weight of the transiently transfected ERK5 kinase domain.
Govarts C et al. Analysis of antibody reactivity in paired cerebrospinal fluid and serum of a relapsing remitting multiple sclerosis patient. Autoimmunity42:699-704 (2009).
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