The proliferation of MC/9 cells treated with increasing concentrations of mGM-CSF was assessed. After 48 hour treatment with mGM-CSF, cells were incubated with a tetrazolium salt and the OD450 - OD650 was determined.
The purity of recombinant mGM-CSF was determined by SDS-PAGE of 6 µg reduced (+) and non-reduced (-) recombinant mGM-CSF and staining overnight with Coomassie Blue.
Western blot analysis of extracts from MC/9 cells untreated or treated with mGM-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 mGM-CSF. Carrier free: Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.2.
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 mGM-CSF. All lots are greater than 98% pure.
Recombinant mGM-CSF contains no "tags" and the nonglycosylated protein has a calculated MW of 14,112.1. DTT-reduced protein migrates as a 22 kDa polypeptide and non-reduced protein migrates as a 19 kDa polypeptide due to intramolecular cystines. Lower mobility and heterogeneity in SDS-PAGE are due to glycosylation. The expected amino-terminal APTRS of recombinant mGM-CSF was verified by amino acid sequencing.
The bioactivity of recombinant mGM-CSF was determined in a MC/9 cell proliferation assay. The ED50 of each lot is between 6-25 pg/ml.
Less than 0.01 ng endotoxin/1μg mGM-CSF.
Recombinant mouse GM-CSF (mGM-CSF) Ala18-Lys141 (Accession #NP_034099) was expressed in human 293 cells at Cell Signaling Technology.
GM-CSF is produced by activated T cells, NK cells and macrophages (1,2). Target cells include granulocytes, monocyte precursors and subsets of differentiated myeloid cells (1,3,4). Many target cells require GM-CSF for survival. GM-CSF induces proliferation, is involved in the 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 (5). 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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