Solid-Phase Metal Chelate Assay for Quantifying Total Protein: Resistance to Chemical Interference

Abstract

Recently, we developed reversible metal chelate stains that are fully compatible with immunoblotting and protein sequencing. Membrane supports are incubated in Ferrozine®/ferrous complex followed by ferrocyanide/ferric complex (double-metal chelate [DMC] stain). Proteins are quantified by computerized densitometry. In this study, the metal chelate stains are used for routine protein quantitation. Manually applying samples to membranes leads to variable spot spreading. Better results are achieved using a slot-blot apparatus to maintain a constant application area. The Ferrozine/ferrous and DMC assays are compared to colloidal gold and bicinchroninic acid (BCA) assays with respect to chemical interference, protein-to-protein variation, dynamic linear range and sensitivity. The DMC assay provides a superior linear range (100-fold) when compared with colloidal gold (10-fold range) and BCA assays (47-fold). Though the colloidal gold assay is more sensitive, it suffers from poor reproducibility, high protein-to-protein variation and lower tolerance to interfering agents. The BCA assay has the least protein-to-protein variation but is also least sensitive and most susceptible to interfering agents.