Direct dye binding--a quantitative assay for solid-phase immobilized protein.

Abstract

A direct dye-binding procedure was established for the quantification of protein after its immobilization on a solid phase, using IgG and BSA as model proteins. The assay, which in the range 0-5 mg protein/ml gel correlates well with indirect protein determination by A280 as well as determination of protein hydrolyzed from the gel, is based on a modified Bradford dye-binding assay. As the protein coupled to the gel binds the dye, a decrease in A465 of the supernatant is measured. Three solid supports commonly used for protein immobilization (Sepharose, Sephadex, Sephacryl) were found to be compatible with the dye-binding assay while nonspecific dye binding was found to HEMA gels. Protein was coupled to Sephacryl S-1000 using three different activation methods (aldehyde, hydrazine, and adipic acid dihydrazide). Artifactual dye-binding was not observed using any of the three different "linkers." The assay is easily carried out and represents a useful tool, e.g., when optimizing procedures for protein immobilization.