FTO, Human, BioAssay(TM) ELISA Kit (Alpha-ketoglutarate-dependent Dioxygenase FTO, Fat Mass and Obesity-associated Protein, 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. FTO is widely expressed in a variety of human tissues, with highest levels in brain and pancreatic islets. Bioinformatics analysis indicates that FTO shares sequence motifs with iron- and 2-oxoglutarate (2OG)-dependent oxygenases. The FTO (human) (IntraCellular) ELISA Kit is to be used for the in vitro quantitative determination of human FTO in cell lysates or cell-based assays (screening). This assay is a sandwich ELISA for quantitative determination of human 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 human FTO. It does cross-react with human adiponectin, human RBP4, human Nampt, human vaspin, human progranulin, human resistin, human clusterin, human GPX3, human sirtuin 1, human IDO, human IL-33, human ANGPTL3, human ANGPTL4, human FGF21, mouse progranulin, mouse ANGPTL3, mouse leptin, rat Nampt. The assay range is 0.156-10ng FTO/ml and a detection limit of 50pg/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 human FTO in cells.\n\nSensitivity:\n50pg/ml \n\nRange:\n0.156-10ng FTO/ml\n\nSpecificity:\nMammalian\n\nTest Principle:\nThis assay is a sandwich ELISA for quantitative determination of human 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\nHuman FTO Standard (lyophilized): 20ng\nHuman 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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