The viability of L-929 cells treated with increasing concentrations of mTNF-α in the presence of 2 ng/ml actinomycin D was assessed. Cells were stained with crystal violet at the end of treatment and the OD595 was determined.
The purity of recombinant mTNF-α was determined by SDS-PAGE of 6 µg reduced (+) and non-reduced (-) recombinant mTNF-α and staining overnight with Coomassie Blue.
Western blot analysis of extracts from HeLa cells, untreated or treated with mouse TNF-α for 20 minutes, 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 20 μg BSA per 1 μg mTNF-α. Carrier free: Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.2.
Stable in lyophilized state at -20°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 mTNF-α. All lots are greater than 98% pure.
Less than 0.01 ng endotoxin/1 μg mTNF-α.
The bioactivity of recombinant mTNF-α was determined in a L-929 cell viability assay. The ED50 of each lot is between 2-8 pg/ml.
Based on amino acid sequencing, greater than 50% of recombinant mTNF-α starts at Leu80 (LRSSS) and has a calculated MW of 17,257. The remainder starts at Ser82 (SSSQN). DTT-reduced and non-reduced protein migrate as 16 kDa polypeptides.
Recombinant mouse TNF-α (mTNF-α) Leu80-Leu235 (Accession #NP_038721) was expressed in human 293 cells at Cell Signaling Technology.
TNF-α, the prototypical member of the TNF protein superfamily, is a homotrimeric type-II membrane protein (1,2). Membrane bound TNF-α is cleaved by the metalloprotease TACE/ADAM17 to generate a soluble homotrimer (2). Both membrane and soluble forms of TNF-α are biologically active. TNF-α is produced by a variety of immune cells including T cells, B cells, NK cells and macrophages (1). Cellular response to TNF-α is mediated through interaction with receptors TNF-R1 and TNF-R2 and results in activation of pathways that favor both cell survival and apoptosis depending on the cell type and biological context. Activation of kinase pathways (including JNK, ERK (p44/42), p38 MAPK and NF-κB) promotes the survival of cells, while TNF-α mediated activation of caspase-8 leads to programmed cell death (1,2). TNF-α plays a key regulatory role in inflammation and host defense against bacterial infection, notably Mycobacterium tuberculosis (3).
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