FTO, Mouse, BioAssay(TM) ELISA Kit (Alpha-ketoglutarate-dependent Dioxygenase FTO, Fat Mass and Obesity-associated Protein, Protein Fatso, Kiaa1752)

FTO, Fat mass-and obesity-associated gene, was discovered as a responsible gene causing the mouse 'fused toes' mutation. The predicted 502aa Fto protein has a calculated molecular mass of 58kD 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 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 450nm 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 0.156-10ng FTO/ml and a detection limit of 20pg/ml (based on adding two standard deviations to the mean value of the (50) zero standards).\n\nSample Type:\nCell lysates.\n\nIntended Use:\nFor the quantitative determination of mouse FTO in cells.\n\nSensitivity:\n20pg/ml \n\nRange:\n0.156-10ng FTO/ml\n\nSpecificity:\nMammalian\n\nTest Principle:\nThis assay is a sandwich 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 450nm after acidification and is directly proportional to the concentration of FTO in the samples.\n\nKit Components:\nPre-coated Microtiter Plate: 12x8 well strips\nWash Buffer (10X): 1x50ml\nDiluent (5X):1x50ml\nLysis Buffer (10X): 1x12ml\nDetection Antibody: 1x12ml\nDetector 100X (HRP conjugated anti-IgG): 1x150ul\nMouse FTO Standard (lyophilized): 20ng\nMouse FTO QC Sample (lyophilized): 1 vial\nSubstrate Solution (TMB): 1x12ml\nStop Solution: 1x12ml\nPlate Sealers: 3 each\n\nStorage and Stability:\nStore powder at 4 degrees C liquid at -20 degrees C. Store other components at 4 degrees C. Stable for at least 6 months For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.

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