The proliferation of M-NFS-60 cells treated with increasing concentrations of hG-CSF was assessed. After 72 hour treatment with hG-CSF, cells were incubated with a tetrazolium salt and the OD450 - OD650 was determined.
The purity of recombinant hG-CSF was determined by SDS-PAGE of 6 µg reduced (+) and non-reduced (-) recombinant hG-CSF and staining overnight with Coomassie Blue.
Western blot analysis of extracts from M-NSF-60 cells untreated or treated with hG-CSF for 15 minutes, using Phospho-Stat3 (Tyr705) (D3A7) Rabbit mAb #9145 (upper) or Stat3 Antibody #9132 (lower).
With carrier: Lyophilized from a 0.22 μm filtered solution of 40 mM phosphate pH 4.0 containing 250 mM NaCl and 20 μg BSA per 1 μg hG-CSF. Carrier free: Lyophilized from a 0.22 μm filtered solution of 40 mM phosphate pH 4.0 containing 250 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 hG-CSF. All lots are greater than 98% pure.
Recombinant hG-CSF contains no "tags" and the nonglycosylated protein has a calculated MW of 18,986. DTT-reduced and non-reduced protein migrate as 18 kDa polypeptides. The expected amino-terminal TPLGP of recombinant hG-CSF was verified by amino acid sequencing.
The bioactivity of recombinant hG-CSF was determined in a M-NFS-60 cell proliferation assay. The ED50 of each lot is between 20-150 pg/ml.
Less than 0.01 ng endotoxin/1μg hG-CSF.
Recombinant human G-CSF (hG-CSF) Thr31-Pro204 (Accession #NP_757373) was expressed in human 293 cells at Cell Signaling Technology.
G-CSF is a hematopoietic cytokine essential for neutrophil development, survival, and egress from bone marrow (1-4). Macrophages and monocytes are the predominant producers of G-CSF (3) and endothelial cells, fibroblasts and neuronal cells can produce G-CSF in response to inflammatory stimuli (3). G-CSF inhibits apoptosis in neutrophils and neurons (4,5). G-CSF stimulates proliferation and differentiation of neuronal progenitor cells (5). G-CSF binding to G-CSFR induces receptor dimerization and activation of Jak1/2 tyrosine phosphorylation (3,6). Signaling is through Stat3, ERK, p38, and Akt (5,6). Absence of functional G-CSF or its receptor in humans and mice causes neutropenia (7,8).
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