The proliferation of MCF 10A cells treated with increasing concentrations of hEpiregulin was assessed. After 24 hr treatment, cells were labeled with BrdU for 4 hr. BrdU incorporation was determined by ELISA and the OD450-OD690 was determined.
The purity of recombinant hEpiregulin was determined by SDS-PAGE of 6 µg reduced (+) and non-reduced (-) recombinant hEpiregulin and staining overnight with Coomassie Blue.
Western blot analysis of extracts from MCF 10A cells, untreated or treated with hEpiregulin for 10 minutes, using Phospho-EGF Receptor (Tyr1173) (53A5) Rabbit mAb #4407 (upper) and EGF Receptor (C74B9) Rabbit mAb #2646 (lower).
With carrier: Lyophilized from a 0.22 μm filtered solution of 20 mM citrate, pH 3.0 containing 100 mM NaCl and 20 μg BSA per 1 μg hEpiregulin. Carrier free: Lyophilized from a 0.22 μm filtered solution of 20 mM citrate, pH 3.0 containing 100 mM NaCl.
Stable in lyophilized state at 4°C for 1 year after receipt. Sterile stock solutions reconstituted with carrier protein are stable at 4°C for 2 months and at -20°C for 6 months. Avoid repeated freeze-thaw cycles.Maintain sterility. Storage at -20°C should be in a manual defrost freezer.
>98% as determined by SDS-PAGE of 6 μg reduced (+) and non-reduced (-) recombinant hEpiregulin. All lots are greater than 98% pure.
Based on amino acid sequencing, greater than 50% of recombinant hEpiregulin has a Met on the amino-terminal Val63 (MVSIT) and has a calculated MW of 5,406. The remainder starts at Val63 (VSITK). DTT-reduced and non-reduced protein migrate as 5 kDa polypeptides.
The bioactivity of recombinant hEpiregulin was determined in a MCF 10A cell proliferation assay. The ED50 of each lot is between 0.10-0.85 ng/ml.
Less than 0.01 ng endotoxin/1 μg hEpiregulin.
Recombinant human Epiregulin (hEpiregulin) Val63-Leu108 (Accession #NP_001423) was produced in E. coli at Cell Signaling Technology.
Epiregulin is a member of the EGF family that is highly expressed in many cancer-specific cells, cells of the placenta and macrophages (1,2). Epiregulin induces keratinocyte and corneal epithelial cell proliferation, oocyte maturation and may function in blastocyst implantation during pregnancy (3-5). Active soluble epiregulin is produced by proteolytic processing of a transmembrane precursor. Epiregulin is the only EGF family member known to bind to both the ErbB1/HER1 and ErbB4/HER4 EGF receptors (6). Epiregulin binding induces receptor homodimerization or heterodimerization with other Erb/Her receptors. Epiregulin activates the ERK1/2, and PI3K/Akt pathways (1,3).
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