Description
Description: This assay employs a two-site sandwich ELISA to quantitate XPO6 in samples. An antibody specific for XPO6 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any XPO6 present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for XPO6 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 XPO6 bound in the initial step. The color development is stopped and the intensity of the color is measured.
Overview: Exportins, such as XPO6, recruit cargo in the nucleoplasm in the presence of RAN-GTP and form ternary export complexes. These complexes are transported through nuclear pore complexes to the cytoplasm, where GTP is hydrolyzed and the export complex is disassembled.
The preferred cargo was the profilin-actin complex, since XPO6 showed little or no binding to either protein in the absence of the other. Analysis of the weak interaction between XPO6 and isolated actin suggested that XPO6 bound the complex via association with the actin component. Knockdown of Drosophila Exp6 by RNA interference abolished nuclear exclusion of actin and resulted in the appearance of nuclear actin paracrystals