Western blot analysis was performed on membrane enriched extracts (30 µg lysate) of MDA-MB-231 (Lane 1), MDA-MB-231 treated with PMA (100ng PMA for 16h) (Lane 2), Caco-2 (Lane 3), A-431 (Lane 4), PC-3 (Lane 5), U-2 OS (Lane 6) and HCT-116 (Lane7).The blots were probed with Amphiregulin Rabbit polyclonal Antibody (Product # PA5-16621, 2 µg/ml) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/ml, 1:2500 dilution). A 25 kDa band corresponding to Amphiregulin was observed across the cell lines tested and was enhanced upon treatment in MDA-MB-231 cell line. Apart from desired band of 25 KDa, ~60 kDa band is seen across the cell lines showing the glycosylated form of the protein. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX), 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 iBlot® 2 Dry Blotting System (Product # IB21001). 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, Mouse|
|Host / Isotype||Rabbit / IgG|
|Immunogen||A synthetic 19-mer peptide, corresponding to aa 26-44 from the secreted amphiregulin of human origin|
|Storage buffer||PBS, pH 7.4, with 0.2% BSA|
|Contains||0.09% sodium azide|
|Storage Conditions||4° C|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||3-5 µg/10^6 cells|
|Immunoprecipitation (IP)||10 µg/ml|
|Western Blot (WB)||2 µg/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.
PA5-16621 targets Amphiregulin in IP and WB applications and shows reactivity with Human and mouse samples.
The PA5-16621 immunogen is a synthetic 19-mer peptide, corresponding to aa 26-44 from the secreted amphiregulin of human origin.
Amphiregulin (AR) binds to EGF-Receptor (EGFR) with lower affinity than EGF. The mature secreted form AR is an 84-amino acid residue glycosylated polypeptide growth regulator, which is generated by proteolytic processing of a 252-amino acid transmembrane precursor. Seven different polypeptide ligands, which derive from distinct genes, are capable of binding to the extracellular domain of EGFR. These ligands include EGF, TGFalpha, AR, HB-EGF, cripto-1, epiregulin, and betacellulin. All of these growth factors contain a characteristic EGF-like domain which is defined by 6 evenly spaced cysteine residues that generate 3 loops through the formation of disulfide bonds. AR protein has been localized by immunohistochemistry to the epithelium of the colon, stomach, pancreas, breast, and placenta. AR is reportedly overexpressed in human cancers of breast, colon, stomach, and pancreas.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.