The viability of L-929 cells treated with increasing amounts of hLT-α in the presence of 2 ng/ml actinomycin D was determined. After 24 hour treatment with hLT-α, cells were incubated with a tetrazolium salt and the OD450 - OD650 was determined.
The purity of recombinant hLT-α was determined by SDS-PAGE of 6 µg reduced (+) and non-reduced (-) recombinant hLT-α and staining overnight with Coomassie Blue.
With carrier: Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.2 containing 20 μg BSA per 1 μg hLT-α. 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 hLT-α. All lots are greater than 98% pure.
Recombinant hLT-α contains no "tags" and the nonglycosylated protein has a calculated MW of 18,548. DTT-reduced and non-reduced protein migrate as 24-31 kDa polypeptides. Lower mobility and heterogeneity in SDS-PAGE are due to glycosylation. The expected amino-terminal PGVGL of recombinant hLT-α was verified by amino acid sequencing.
The bioactivity of recombinant hLT-α was determined in an L-929 cell viability assay. The ED50 of each lot is between 15-150 pg/ml.
Less than 0.01 ng endotoxin/1 μg hLT-α.
Recombinant human LT-α (hLT-α) Pro36-Leu205 (Accession #NP_000586) was expressed in human 293 cells at Cell Signaling Technology.
Lymphotoxin-α (LT-α), also known as TNF–β, is a member of the TNF superfamily of proteins (1). NK cells, T cells, B cells, and lymphoid tissue-inducer cells express LT-α (1). LT-α can be secreted as a soluble homotrimer or form membrane bound heterotrimers with lymphotoxin-β (LT α1β2 or LT α2β1) which can be cleaved from the cell surface by matrix metalloproteases (1,2). Soluble LT-α binds to and signals through TNFR1/TNFR2, activating the canonical NF-κB pathway (1). In contrast, LT α1β2 heterodimers bind to the LTβR receptor and activate the noncanonical NF-κB pathway (1). As a result, LT-α and TNF-α have overlapping functions. Soluble LT-α and LT α1β2 play key roles in lymphangiogenesis (3). The LT α1β2/LTβR axis is essential for the development of lymphoid tissue (1,3).
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