The proliferation of FLT3 transfected BaF3 cells treated with increasing concentrations of hFLT3L was assessed. After 48 hour treatment with hFLT3L, cells were incubated with a tetrazolium salt and the OD450 - OD650 was determined.
The purity of recombinant hFLT3L was determined by SDS-PAGE of 6 µg reduced (+) and non-reduced (-) recombinant hFLT3L and staining overnight with Coomassie Blue.
Western blot analysis of extracts from BaF3/FLT3 cells untreated or treated with hFLT3L for 15 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 hFLT3L. 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 hFLT3L. All lots are greater than 98% pure.
Recombinant hFLT3L contains no "tags" and the nonglycosylated protein has a calculated MW of 18,037. DTT-reduced and non-reduced protein migrate as 24 kDa polypeptides due to glycosylation, with non-reduced having slightly greater mobility due to intramolecular cystines. The expected amino-terminal TQDCS of recombinant hFLT3L was verified by amino acid sequencing.
The bioactivity of recombinant hFLT3L was determined in a cell proliferation assay using BAF3 cells transfected with FLT3. The ED50 of each lot is between 1.1 - 2.3 ng/ml.
Less than 0.01 ng endotoxin/1 μg hFLT3L.
Recombinant human FLT3L (hFLT3L) Thr27-Pro185 (Accession #BC126293.1) was expressed in human 293 cells at Cell Signaling Technology.
FLT3L is produced by T cells and stromal fibroblasts (1,2). FLT3L targets many cell types including hematopoietic stem cells, B cells, T cells, dendritic cells, and NK cells (1,2). FLT3L in combination with other factors stimulates differentiation and proliferation of hematopoietic multipotent progenitors and promotes proliferation of NK cells and dendritic cell subsets. There are two splice variants of FLT3L. Both the integral membrane and soluble splice variants are biologically active (1). Binding of FLT3L to its cognate tyrosine kinase receptor, FLT3, activates STAT3 and STAT5 (1,3). FLT3L is being tested for its ability to stimulate anti-tumor immune response via stimulation of dendritic and NK cells.
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