Description
Description: This assay employs a two-site sandwich ELISA to quantitate ZFYVE27 in samples. An antibody specific for ZFYVE27 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any ZFYVE27 present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for ZFYVE27 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 ZFYVE27 bound in the initial step. The color development is stopped and the intensity of the color is measured.
Overview: Transient transfection of HeLa cells with epitope-tagged ZFYVE27 constructs revealed that ZFYVE27 is predominantly expressed in punctate vesicles, although a tubular pattern of expression was observed in a small proportion of cells.
Colocalization studies detected overlapping expression with the endosomal marker EEA1 and the endoplasmic reticulum marker RTN1. Coimmunoprecipitation studies in NIH3T3 cells determined that spastin mediates interaction with ZFYVE27 through its N-terminal domain, which consists of an MIT (microtubule-interacting and trafficking) motif. An immunoprecipitation assay confirmed the interaction of endogenous spastin with ZFYVE27