Western blot analysis of MAP2K1 [pSpS217/221] was performed by loading 20 µg of A431 (lane1) and Jurkat (lane2) cell lysates using Novex® NuPAGE® 4-12 % Bis-Tris gel (NP0321BOX), XCell SureLock™ Electrophoresis System (EI0002), Novex® Sharp Pre-Stained Protein Standard (LC5800), and iBlot® Dry Blotting System (IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. MAP2K1 [pSpS217/221] was detected at ~44 kDa using ABfinity™ MAP2K1 [pSpS217/221] Recombinant Rabbit Monoclonal Antibody (701267) at 1-3 µg/ml in 2.5 % skim milk at 4°C overnight on a rocking platform. To confirm specificity, the corresponding blot on right was incubated with Lambda phosphatase and its reactivity with antibody was tested. Goat Anti-Rabbit IgG-HRP Secondary Antibody (G21234) at 1:5000 dilution was used and chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (WP20005).
|Tested species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Phosphopeptide corresponding to amino acids 212–226 of human MAP2K1 [pSpS217/221]|
|Contains||0.09% sodium azide|
|Storage Conditions||Maintain refrigerated at 2-8°C for up to 1 month. For long term storage store at -20°C|
|Tested Applications||Dilution *|
|ChIP assay (ChIP)||1 ul|
|Western Blot (WB)||1-3µg/ml|
* 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.
This antibody is predicted to react with mouse, rat, Drosophila, non-human primate and rabbit based on sequence homology.
ABfinity™ recombinant antibodies are rabbit monoclonal antibodies, unmatched for producing superior results. ABfinity™ antibodies are developed by immunizing animals, screening for functionality, cloning the immunogen-specific antibody genes into high-level mammalian expression vectors, produced on a large scale, and purified with Protein A.
ABfinity™ monoclonal antibodies resemble rabbit monoclonals isolated from serum or produced by hybridomas, but demonstrate greater specificity and sensitivity. Because ABfinity™ recombinant antibodies are derived from cloned DNA sequences of the heavy and light antibody chains, they are not susceptible to cell-line drift or lot-to-lot variation, thus allowing for peak specificity and performance.
Intact IgG appears on a non-reducing gel as ~150 kDa band and upon reduction generating a ~25 kDa light chain band and a ~50 kDa heavy chain.
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.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.