The proliferation of MCF-10A cells treated with increasing concentrations of hTGF-α was assessed. After 24 hour treatment, cells were labeled with BrdU for 4 hours. BrdU incorporation was determined by ELISA and the OD450-OD650 was determined.
The purity of recombinant hTGF-α was determined by SDS-PAGE of 6 µg reduced (+) and non-reduced (-) recombinant hTGF-α and staining overnight with Coomassie Blue.
Western blot analysis of extracts from HeLa cells untreated or treated with hTGF-α for 10 minutes, using Phospho-EGF Receptor (Tyr1173) (53A5) Rabbit mAb #4407 (upper) and EGF Receptor Antibody #2232 (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 hTGF-α. 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 hTGF-α. All lots are greater than 98% pure.
Based on amino acid sequencing, greater than 50% of recombinant hTGF-α has a Met on the amino-terminal Val40 (MVVSH) and has a calculated MW of 5,683. The remainder starts at Val40 (VVSHF). DTT-reduced and non-reduced protein migrate as 5 kDa polypeptides.
The bioactivity of recombinant hTGF-α was determined in a MCF-10A cell proliferation assay. The ED50 of each lot is between 0.25-1.0 ng/ml.
Less than 0.01 ng endotoxin/1 μg hTGF-α.
Recombinant human TGF-α (hTGF-α) Val40-Ala89 (Accession #NP_003227) was produced in E. coli at Cell Signaling Technology.
TGF-α is a member of the EGF family of proteins and is expressed by epithelial cells, keratinocytes, macrophages, and many tumor-derived cell lines (1-4). Soluble TGF-α is produced by proteolytic cleavage of a transmembrane precursor (5). TGF-α promotes cell proliferation and differentiation of a diverse array of cell types including cancer and neural cells (1,4,6). TGF-α binds to EGFR/ErbB1 and induces receptor dimerization and tyrosine kinase activation (7). TGF-α activates the MAPK pathways and involves Stat3 and Stat5 (1,6). Increased levels of TGF-α expression are associated with many cancers including breast cancer (1,8,9).
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