| Description |
FTO, Fat mass-and obesity-associated gene, was discovered as a responsible gene causing the mouse ‘fused toes’ mutation. The predicted 502-amino acid Fto protein has a calculated molecular mass of 58 kD and contains an N-terminal bipartite nuclear localization signal. RT-PCR detected Fto expression throughout mouse embryonic development and in all adult mouse tissues examined except heart and skin. Bioinformatics analysis indicates that FTO shares sequence motifs with iron- and 2-oxoglutarate (2OG)-dependent oxygenases. Recombinant murine Fto catalyzes the iron- and 2OG-dependent demethylation of 3-methylthymine in single-stranded DNA, with concomitant production of succinate, formaldehyde, and carbon dioxide. In wildtype mice, Fto mRNA is most abundant in the brain, particularly in hypothalamic nuclei, and are regulated by feeding and fasting. This assay is a sandwich Enzyme Linked-Immunosorbent Assay (ELISA) for quantitative determination of mouse FTO in cells. A monoclonal antibody specific for FTO has been precoated onto the 96-well microtiter plate. Standards and samples are pipetted into the wells for binding to the coated antibody. After extensive washing to remove unbound compounds, FTO is recognized by the addition of a purified polyclonal antibody specific for FTO (Detection Antibody). After removal of excess polyclonal antibody, HRP conjugated anti-rabbit IgG (Detector) is added. Following a final washing, peroxidase activity is quantified using the substrate 3,3’,5,5’-tetramethylbenzidine (TMB). The intensity of the color reaction is measured at 450 nm after acidification and is directly proportional to the concentration of FTO in the samples. This ELISA is specific for the measurement of natural and recombinant mouse FTO. It does cross-react with mouse adiponectin, mouse RBP4, mouse Nampt, mouse vaspin, mouse progranulin, mouse resistin, mouse clusterin, mouse GPX3, mouse CD137, rat adiponectin, rat Nampt, human adiponectin, human Nampt, human progranulin. The assay range is 0.156 – 10 ng FTO/ml and a detection limit of 20 pg/ml (based on adding two standard deviations to the mean value of the (50) zero standards).
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