The purity of recombinant mIGF-I was determined by SDS-PAGE of 6 µg reduced (+) and non-reduced (-) recombinant mIGF-I and staining overnight with Coomassie Blue.
The ability of mIGF-I to induce phosphorylation of Akt was assessed. After serum starvation, NIH/3T3 cells were treated with increasing concentrations of mIGF-I for 10 minutes. Cells were lysed, and phospho-Akt was quantified using PathScan® Phospho-Akt (Thr308) Sandwich ELISA Kit #7252. OD450 is shown.
The proliferation of primary human dermal fibroblasts treated with increasing concentrations of mIGF-I was assessed. After 72 hour treatment with mIGF-I, cells were incubated with a tetrazolium salt and the OD450 - OD650 was determined.
Western blot analysis of extracts from NIH/3T3 cells, untreated or treated with increasing concentrations of mIGF-I for 10 minutes, using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060 (upper) and Akt1 (C73H10) Rabbit mAb #2938 (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 mIGF-I. 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 mIGF-I. All lots are greater than 98% pure.
Recombinant mIGF-I has a Met on the amino terminus and has a calculated MW of 7,808. DTT-reduced and non-reduced protein migrate as 6 kDa polypeptides. The expected amino-terminal MGPET of recombinant mIGF-I was verified by amino acid sequencing.
The bioactivity of recombinant mIGF-I was determined in a cell proliferation assay using primary human dermal fibroblasts. The ED50 of each lot is between 3-15 ng/ml.
Less than 0.01 ng endotoxin/1 μg mIGF-I.
Recombinant mouse IGF-I (mIGF-I) Gly33-Ala102 (Accession #NP_001104746) was produced in E. coli at Cell Signaling Technology.
Most circulating endocrine-acting IGF-I is produced by hepatocytes, and paracrine- or autocrine-acting IGF-I is produced by defined cell types within specific tissues (1,2). Many neoplastic cells produce IGF-I, which regulates a number of cellular processes including energy metabolism, proliferation, and cell survival (3,4). IGF-I activity is regulated by one or more of the six extracellular IGF-binding proteins (IGFBPs). IGFBPs bind to IGF-I, and most inhibit the binding between IGF-I and the IGF-I receptor (IGFIR) (1,2). Some IGFBPs may increase cell responses to IGF-I. Binding of IGF-I to IGFIR activates the Akt, JNK, and Erk pathways (2). IGF-I and IGFIR are frequently expressed by cancer cells and may contribute to the proliferation and viability of a number of cancer types (1,2).
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