The ability of mHis6IL-6 to induce phosphorylation of Stat3 was assessed. B9 cells were treated with increasing concentrations of mHis6IL-6 for 10 min. Cells were lysed, and phospho-Stat3 was quantified using PathScan® Phospho-Stat3 (Tyr705) Sandwich ELISA Kit #7300. OD450 is shown.
The proliferation of B9 cells treated with increasing concentrations of mHis6IL-6 was assessed. After 48 hr treatment with mHis6IL-6, cells were incubated with a tetrazolium salt and the OD450 - OD650 was determined.
The purity of recombinant mHis6IL-6 was determined by SDS-PAGE of 6 µg reduced (+) and non-reduced (-) recombinant mHis6IL-6 and staining overnight with Coomassie Blue.
Western blot analysis of extracts from B9 cells, untreated or treated with increasing concentrations of mHis6IL-6 for 10 min, using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb #9145 (upper) and Stat3 (79D7) Rabbit mAb #4904 (lower).
With carrier: Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.2 containing 20 μg BSA per 1 μg mHis6IL-6. 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 mHis6IL-6. All lots are greater than 98% pure.
Recombinant N-terminally His6-tagged mIL-6 has a calculated MW of 24,311. DTT-reduced and non-reduced protein migrate as 31-36 kDa polypeptides. Lower mobility and heterogeneity in SDS-PAGE are due to glycosylation. The expected amino terminus of recombinant mHis6IL-6 was verified by amino acid sequencing.
The bioactivity of recombinant mIL-6 was determined in a B9 cell proliferation assay. The ED50 of each lot is between 0.5 and 5 pg/ml.
Less than 0.01 ng endotoxin/1 μg mHis6IL-6.
Recombinant mouse His6IL-6 (mHis6IL-6) Phe25-Thr211 (Accession #NP_112445) was expressed in human 293 cells at Cell Signaling Technology.
IL-6 is a potent inducer of the acute phase response and is produced by T cells, macrophages, fibroblasts, endothelial, and other cells (1,2). IL-6 induces proliferation and differentiation and acts on B cells, T cells, thymocytes, among others. IL-6 in concert with TGF-β is important for developing Th17 responses. IL-6 binds to IL-6Rα and through this association induces gp130 homodimerization (1). gp130 homodimerization triggers the Jak/Stat cascade and the SHP2/MAPK (Erk) cascade (1,3,4). IL-6 also forms a complex with an IL-6Rα splice variant that is non-membrane associated (3). This IL-6/soluble IL-6Rα complex can activate the gp130 signaling pathway on cells that express gp130, but not IL-6Rα (3). Research studies have shown that IL-6 may contribute to metastatic breast cancer by increasing expression of proangiogenic VEGF (5).
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