Characterization of a monoclonal antibody capable of reliably quantifying expression of human Copper Transporter 1...

Human copper transporter 1 (hCTR1) is the high-affinity copper influx transporter in mammalian cells that also mediates the influx of cisplatin. Loss of hCTR1 expression has been implicated in the development of resistance to this cancer chemotherapeutic agent. It has turned out to be very difficult to develop antibodies to hCTR1 and polyclonal antibodies produced by different laboratories have yielded conflicting results. We have characterized a newly-available rabbit monoclonal antibody that reacts with an epitope on the N-terminal end of hCTR1 that now permits rigorous identification and quantification of hCTR1 using Western blot analysis. Postnuclear membrane (PNM) preparations made from cells engineered to express high levels of myc-tagged hCTR1, and cells in which the expression of hCTR1 was knocked down, were used to characterize the antibody. The identity of the bands detected was confirmed by immunoprecipitation, surface biotinylation and deglycosylation of myc-tagged hCTR1. Despite the specificity expected of a monoclonal antibody, the anti-hCTR1 detected a variety of bands in whole cell lysates (WCL), which made it difficult to quantify hCTR1. This problem was overcome by isolating post-nuclear membranes and using these for further analysis. Three bands were identified using this antibody in PNM preparations that migrated at 28, 33-35 and 62-64kDa. Multiple lines of evidence presented here suggest that the 33-35 and 62-64kDa bands are hCTR1 whereas the 28kDa band is a cross-reacting protein of unknown identify. The 33-35kDa band is consistent with the expected MW of the glycosylated hCTR1 monomer. This analysis now permits rigorous identification and quantification of hCTR1.