The production of human IL-8 by primary human fibroblasts cultured with increasing concentrations of hIL-1α was assessed. Media from cells incubated with hIL-1α for 24 hours was collected and assayed for IL-8 by ELISA and the OD450-OD650 was determined.
The purity of recombinant hIL-1α was determined by SDS-PAGE of 6 µg reduced (+) and non-reduced (-) recombinant hIL-1α and staining overnight with Coomassie Blue.
Western blot analysis of extracts from primary human fibroblasts, untreated or treated with hIL-1α for 15 minutes, using Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb #3033 (upper) and NF-κB p65 Antibody #3034 (lower).
With carrier: Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.2 containing 20 μg BSA per 1 μg hIL-1α. 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 hIL-1α. All lots are greater than 98% pure.
Recombinant hIL-1α contains no "tags" and the nonglycosylated protein has a calculated MW of 18,063. DTT-reduced and non-reduced protein migrate as 22 kDa polypeptides due to glycosylation. The expected amino-terminal SSPFS of recombinant hIL-1α was verified by amino acid sequencing.
The bioactivity of recombinant hIL-1α was determined by its ability to induce IL-8 production by primary human fibroblasts. The ED50 of each lot is between 0.6-2.0 ng/ml.
Less than 0.01 ng endotoxin/1 μg hIL-1α.
Recombinant human IL-1α (hIL-1α) Ser113-Ala271 (Accession #X03833) was expressed in human 293 cells at Cell Signaling Technology.
IL-1α is a pro-inflammatory cytokine produced by activated monocytes, lymphocytes and epithelial cells (1). IL-1α is synthesized as an active precursor protein that appears to be cleaved by cytosolic proteases into its mature form (1,2). Often, precursor and mature forms of IL-1α are primarily retained intracellularly rather than constitutively secreted. (1,2). Signaling by IL-1α involves IL-1α binding to an IL-1 accessory protein (IL-1-AcP) and then the complex binds to IL-1RI (1,2). Signaling is through activation of MAP kinase and NFκB pathways (1,2). IL-1α also binds to an IL-RII that lacks an intracellular signaling domain and thereby serves as a high affinity decoy receptor. Inhibition of IL-1α activity is through IL-1R antagonist (IL-1Ra) that binds IL-1R1 but does not signal. IL-1α has been shown to be a key mediator of virus-induced inflammatory responses in mice (3).
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