Description: This assay employs a two-site sandwich ELISA to quantitate FAM76B in samples. An antibody specific for FAM76B has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any FAM76B present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for FAM76B is added to the wells. After washing, Streptavidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of FAM76B bound in the initial step. The color development is stopped and the intensity of the color is measured.
Overview: FAM76B, Belongs to the FAM76 family. FAM76A and FAM76B pair of paralogues. A sequence alignment of these two proteins highlighted their high degree of conservation, except in the region containing the His-repeat. As hypothesized, the paralogue without the His-tract, FAM76A, presented a diffuse nucleoplasmic staining, while the protein with the polyHis segment, FAM76B, accumulated in nuclear speckles. Similar results were obtained for other pairs of paralogous proteins such as DYRK1A/DYRK1B or POU4F2/POU4F3. Thus, these findings further indicate that the His-repeats in these proteins are necessary for their localization to nuclear speckles