The proliferation of TF-1 cells treated with increasing concentrations of hEPO was assessed. After 48 hour treatment with hEPO, cells were incubated with a tetrazolium salt and the OD450 - OD650 was determined.
The purity of recombinant hEPO was determined by SDS-PAGE of 6 µg reduced (+) and non-reduced (-) recombinant hEPO and staining overnight with Coomassie Blue.
With carrier: Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.2 containing 20 μg BSA per 1 μg hEPO. Carrier free: Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.2.
Stable in lyophilized state at -20°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 hEPO. All lots are greater than 98% pure.
Less than 0.01 ng endotoxin/1μg hEPO.
The bioactivity of recombinant hEPO was determined in a TF-1 cell proliferation assay. The ED50 of each lot is between 50-400 pg/ml.
Recombinant hEPO contains no "tags" and the nonglycosylated protein has a calculated MW of 20,417. DTT-reduced and non-reduced protein migrate as 34-40 kDa polypeptides. Lower mobility in SDS-PAGE is due to glycosylation. The expected amino-terminal APPRL of recombinant hEPO was verified by amino acid sequencing.
Recombinant human Erythropoietin (hEPO) Ala28-Arg193 (Accession #NP_000790) was expressed in human 293 cells at Cell Signaling Technology.
EPO is a 34 kDa protein that is required for the survival and terminal differentiation of erythrocytes (1). EPO is produced by a number of cell types including tubular endothelial cells, interstitial cells, hepatocytes, Kupffer cells, astrocytes, and Schwann cells (2-4). EPO inhibits apoptosis and may protect neuronal cells from death during ischemia and/or neurodegenerative diseases (3,4). EPO inhibits the production of pro-inflammatory cytokines through inhibition of NF-κB signaling (5). EPO protected against the development of diabetes in an animal model by promoting pancreatic β cell survival and growth (6). Binding of EPO to its cognate receptor, EPOR, induces activation of Jak2, Stat5 and Akt (3-5).
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