The proliferation of TF-1 cells treated with increasing concentrations of hGM-CSF was assessed. After 48 hour treatment with hGM-CSF, cells were incubated with a tetrazolium salt and the OD450 - OD650 was determined.
The purity of recombinant hGM-CSF was determined by SDS-PAGE of 6 µg reduced (+) and non-reduced (-) recombinant hGM-CSF and staining overnight with Coomassie Blue.
Western blot analysis of extracts from TF-1 cells, untreated or treated with hGM-CSF for 10 minutes, using Phospho-Stat5 (Tyr694) (C11C5) Rabbit mAb #9359 (upper) and Stat5 (3H7) Rabbit mAb #9358 (lower).
With carrier: Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.2 containing 20 μg BSA per 1 μg hGM-CSF. 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 hGM-CSF. All lots are greater than 98% pure.
Less than 0.01 ng endotoxin/1μg hGM-CSF.
The bioactivity of recombinant hGM-CSF was determined in a TF-1 cell proliferation assay. The ED50 of each lot is between 5-500 pg/ml.
Recombinant hGM-CSF does not have a Met on the amino terminus and has a calculated MW of 14477. DTT-reduced protein migrates as a 14 kDa polypeptide and non-reduced protein has slightly greater mobility due to intramolecular cystines. The expected amino-terminal APARS of recombinant hGM-CSF was verified by amino acid sequencing.
Recombinant human GM-CSF (hGM-CSF) Ala18 - Glu144 (Accession # NM_000758) was produced in E. coli at Cell Signaling Technology.
GM-CSF is produced by activated T cells, NK cells and macrophages (1,5). Target cells include granulocyte, monocyte precursors and subsets of differentiated myeloid cells (1,2,3). Many target cells require GM-CSF for survival. GM-CSF induces proliferation, is involved in hematopoietic differentiation of dendritic cells and is a key factor in differentiation pathways leading from stem cells. GM-CSF activates effector functions of myeloid cells, thereby linking adaptive and innate immunity and in turn may boost anti-tumor immunity (4). GM-CSF receptor is composed of GM-CSFRα and the common β chain, βC, which is also utilized by IL-3 and IL-5 (1). Binding of GM-CSF initiates the Jak2, Stat5 and PI3K/Akt pathways (1).
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