Quantitation of proteins by dot-immunobinding assay. A comparison of visualization methods using eukaryotic...

Various visualization methods were compared for quantitation of proteins by the dot-immunobinding assay. Comparisons were carried out using a multi-subunit protein, eukaryotic initiation factor 2, and monospecific antibodies directed against two of the factor's subunits. The protein was spotted onto nitrocellulose and the membranes were incubated with primary antibody. The antigen-antibody complex was visualized by one of six methods using either alkaline phosphatase-, horseradish peroxidase-, or glucose oxidase-conjugated IgG, or colloidal gold-labelled IgG, colloidal gold-labelled IgG with silver enhancement, or 125I-labelled protein A. The amount of secondary antibody bound was quantitated by densitometric scanning of the nitrocellulose membrane after staining or autoradiography. The sensitivity of each of the methods was similar; each of the visualization methods could detect less than 1 ng of protein by the dot-immunobinding assay. Curves of protein concentration vs. densitometric absorbance were found to fit a parabolic relationship (r2 = 0.99) over a wide range of concentrations.