The inhibition of IL-4 induced proliferation in HT-2 cells treated with increasing concentrations of mTGF-β1 was assessed. After 48 hour treatment with mTGF-β1, cells were incubated with a tetrazolium salt and the OD450 - OD650 was determined.
The purity of recombinant mTGF-β1 was determined by SDS-PAGE of 6 µg reduced (+) and non-reduced (-) recombinant mTGF-β1 and staining overnight with Coomassie Blue.
Western blot analysis of extracts from HT-2 cells, untreated or treated with the mTGF-β1 for 20 minutes, using Phospho-Smad2 (Ser465/467) (138D4) Rabbit mAb #3108 (upper) and Smad2 (D43B4) XP® Rabbit mAb #5339 (lower).
With carrier: Lyophilized from a 0.22 μm filtered solution of 20 mM citrate, pH 3.0 containing 100 mM NaCl and 20 μg BSA per 1 μg mTGF-β1. Carrier free: Lyophilized from a 0.22 μm filtered solution of 20 mM citrate, pH 3.0 containing 100 mM NaCl.
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 mTGF-β1. All lots are greater than 98% pure.
Recombinant mTGF-β1 contains no "tags" and the nonglycosylated protein has a calculated MW of 12,810. DTT-reduced protein migrates as a 13 kDa polypeptide and the non-reduced cystine-linked homodimer migrates as a 25 kDa protein. The expected amino-terminal ALDTN of recombinant mTGF-β1 was verified by amino acid sequencing.
The bioactivity of recombinant mTGF-β1 was determined by assessing inhibition of IL-4 induced HT-2 cell proliferation. The ED50 of each lot is between 40-200 pg/ml.
Less than 0.01 ng endotoxin/1 μg mTGF-β1.
Recombinant mouse TGF-β1 (hTGF-β1) Ala279-Ser390 (Accession #NP_035707) was expressed in human 293 cells at Cell Signaling Technology.
TGF-β1 activities include proliferation, angiogenesis, and promotion or inhibition of many immune events (1-3). TGF-β1 is produced by a number of cell types including regulatory T cells, fibroblasts, epithelial cells, and endothelial cells (3). TGF-β1 binds to TβRII homodimer, which then complexes with TβRI homodimer (1,4). The oligomeric receptor complex phosphorylates subsets of the Smad proteins that then act to induce and repress a number of target genes (1,3,4). TGF-β1 binding can also activate the Erk2, p38, and JNK pathways via TAK1 (4). TGF-β1 appears to promote late stage progression and metastasis in some cancers (1,2).
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