The bioactivity of recombinant hIFN-γ was determined in a virus protection assay. A549 cells were pretreated with increasing concentrations of hIFN-γ (started at 2.5 ng/ml). Cells were then innoculated with encephalomyocarditis virus (EMCV). The OD595 was determined for the surviving cells.
The purity of recombinant hIFN-γ was determined by SDS-PAGE of 1.5 µg reduced (+) and non-reduced (-) recombinant hIFN-γ and staining overnight with Coomassie Blue.
Western blot analysis of extracts from HeLa cells, untreated or treated with hIFN-γ for 20 minutes, using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb #9145 (upper) and Stat3 (124H6) Mouse mAb #9139 (lower).
Working concentration of hIFN-γ generally ranges from 0.1-10 ng/ml.
Recombinant human IFN-γ is supplied as lyophilized material that is very stable at -20°C. It is recommended to reconstitute with sterile water at a concentration of 0.1 mg/ml which can be further diluted in aqueous solutions as needed. Addition of a carrier protein (0.1% HSA or BSA) is recommended for long term storage.
The bioactivity of hIFN-γ was determined in a virus protection assay. The ED50 of each lot is between 0.30-1.2 ng/ml.
Recombinant human IFN-γ was expressed in E. coli and is supplied in a lyophilized form. A greater than 95% purity was determined by SDS-PAGE. Endotoxin levels are less than or equal to 1 EU / 1 μg hIFN-γ.
IFN-γ plays key roles in both the innate and adaptive immune response. IFN-γ activates the cytotoxic activity of innate immune cells such as macrophages and NK cells (1,2). IFN-γ production by NK cells and antigen-presenting cells (APCs) promotes the cell-mediated adaptive immunity by inducing IFN-γ production by T lymphocytes, increasing expression of class I and class II MHC, and enhancing peptide antigen presentation (1). The anti-viral activity of IFN-γ is due to its induction of PKR and other regulatory proteins. Binding of IFN-γ to the IFNGR1/IFNGR2 complex promotes dimerization of the receptor complexes. Binding induces a conformational change in receptor intracellular domains and signaling involves Jak1, Jak2 and Stat1 (3). The critical role of IFN-γ in amplification of immune surveillance and function is supported by increased susceptibility to pathogen infection in IFN-γ or IFNGR knockout mice and in humans with inactivating mutations in IFNGR1 or IFNGR2. IFN-γ also appears to have a role in atherosclerosis (4).
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