Western blot analysis was performed on whole cell extracts (30 ug lysate) of A549 (Lane 1), A549 Serum Starved (Lane 2), A549 treated for 10 minutes with 200 ng/ml of EGF (Lane 3), MCF7 (Lane 4), MCF7 Serum Starved (Lane 5), MCF7 treated for 10 minutes with 200 ng/ml of EGF (Lane 6), MDA-MB-231 (Lane 7), MDA-MB-231 Serum Starved (Lane 8), MDA-MB-231 treated for 10 minutes with 200 ng/ml of EGF (Lane 9). The blots were probed with Anti-HER-2 [pY1248] Rabbit Polyclonal Antibody (Product# 44904G, 1:500-1:2000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP conjugate (Product # G21234,1:5000 dilution). A 185 kDa band corresponding to HER-2 [pY1248] was observed upon treatment across the cell lines tested except in MCF7. An additional band at ~ 55 kDa was observed in MDA-MB-231. Known quantity of protein samples were electrophoresed using Novex® NuPAGE®10 % Bis-Tris gel (Product # NP0301BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with overnight wet transfer system. The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).
|Tested species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||The antiserum was produced against a chemically synthesized phosphopeptide derived from a region of human ErbB-2 that contains tyrosine 1248. The sequence is conserved in rat, hamster and dog.|
|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 *|
|Flow Cytometry (Flow)||3-5 µg/million cells|
|Western Blot (WB)||1:500-1:2000|
* 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.
ErbB2 (also known as Her2 or neu) is a 185 kDa transmembrane receptor tyrosine kinase from the EGFR family that acts to regulate a variety of biological responses including cell growth, differentiation, and tissue development. Ligand binding to the extracellular domain, or overexpression of the receptor leads to phosphorylation on several tyrosine residues within the cytoplasmic domain, and activation. Overexpression or abnormal activation of ErbB2 has been found in a variety of tumors including brain, breast, lung and skin cancer. Autophosphorylation of tyrosine 1139 on ErbB2 allows binding of Grb2 and the Src SH2 domain, which allows activation of the Ras, Raf, ERK1 and 2 and JAK/STAT signaling pathways, respectively.
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