The proliferation of mouse splenic B cells treated with increasing concentrations of hHis6BAFF in the presence of 10 μg/ml of goat anti-mouse IgM μ chain was assessed. After 72 hour treatment with hHis6BAFF, cells were incubated with a tetrazolium salt and the OD450-OD650 was determined.
The purity of recombinant hHis6BAFF was determined by SDS-PAGE of 6 µg reduced (+) and non-reduced (-) recombinant hHis6BAFF and staining overnight with Coomassie Blue.
Western blot analysis of extracts from mouse splenic B cells, untreated or treated with hHis6BAFF for 24 hours, using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370 (upper) and p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb #4695 (lower).
With carrier: Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.2 containing 10 mM DTT and 20 μg BSA per 1 μg hHis6BAFF. Cystines are not required for bioactivity. Carrier free: Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.2 containing 10 mM DTT. Cystines are not required for bioactivity.
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 hHis6BAFF. All lots are greater than 98% pure.
Recombinant N-terminally His6-tagged hBAFF has a calculated MW of 18,066. DTT-reduced and non-reduced protein migrate as 21 kDa polypeptides. The expected amino terminus of recombinant hHis6BAFF was verified by amino acid sequencing.
The bioactivity of recombinant hHis6BAFF was determined in a cell proliferation assay using mouse splenic B cells. The ED50 of each lot is between 0.5-2 ng/ml.
Less than 0.01 ng endotoxin/1 μg hHis6BAFF.
Recombinant human His6BAFF (hHis6BAFF) Ala134-Leu285 (Accession #NP_006564) was expressed in human 293 cells at Cell Signaling Technology.
BAFF, a member of the TNF superfamily of proteins, is a homotrimeric transmembrane protein, which is cleaved to produce a soluble cytokine (1). BAFF may also further oligomerize into 60-mer structures (1). BAFF is expressed by neutrophils, macrophages, dendritic cells, activated T cells, and epithelial cells (1,2). BAFF plays a key role in B cell development, survival, and activation (1,3,4). BAFF binds to three distinct receptors, BAFF-R, TACI, and BCMA (1). These receptors are differentially expressed during B cell development and among B cell subsets (1,2,4). While BAFF-R and BCMA bind to the homotrimeric form of BAFF, TACI only binds to membrane-bound or higher order BAFF structures (1). The BAFF/ BAFF-R interaction activates both canonical and non-canonical NF-κB pathways, PI3K/Akt, and mTor signaling (2,4). Activation of the noncanonical NF-κB pathway via BAFF-R is negatively regulated by TRAF3 (5). Elevated levels of BAFF may exacerbate many autoimmune disorders, making it an attractive therapeutic target (2).
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