Traditional immunoblot assays for tissue concentrations of transferrin receptor (TfR), transferrin (Tf), and ferritin are limited in quantification ability by the functioning of the scanning densitometer. We sought to develop more precise methods of quantifying these proteins of iron metabolism in tissue. An ELISA method using serial dilutions of purified protein antigens or serial dilutions of dialyzed tissue homogenates to coat the ELISA plate was constructed. TfR antigen was purified from iron deficient rat liver via immunoprecipitation and PAGE electrophoresis. Rat Tf and ferritin were obtained commercially. Monoclonal and polyclonal primary antibodies were both used successfully. The secondary antibody was a commercially available APase-conjugated anti-IgG. A linear dose response region determined within each tissue serial dilution allows 2-3 estimates of concentration. Within plate reproducibility is <5% when iron protein concentrations are determined in either rat liver or brain. Importantly, between plate reproducibility is also <5%. Sensitivity was equal to that of immunoblots. This approach thus provides a more reliable approach to quantifying tissue iron proteins.
|Original language||English (US)|
|State||Published - Mar 20 1998|
All Science Journal Classification (ASJC) codes
- Agricultural and Biological Sciences (miscellaneous)
- Biochemistry, Genetics and Molecular Biology(all)
- Cell Biology