Quantitation of related proteins by Western blot analysis

A spectrophotometric assay for quantitation of related proteins following Western blot analysis is described. Proteins were separated by polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose. In the system utilized in this study, immobilized interleukin-1 (IL-1) proteins were identified by ELISA reaction using a rabbit antibody specific for IL-1 beta. Subsequently, the membrane was probed with goat anti-rabbit antibody conjugated to horseradish peroxidase and IL-1 proteins were detected by incubation with 4-chloro-1-naphthol and hydrogen peroxide. The stained IL-1 bands were cut out, exposed to the peroxidase chromogenic substrate, o-phenylenediamine (OPD), and hydrogen peroxide and the rate of reaction was determined spectrophotometrically at 490 nM. A linear relationship between enzyme activity and IL-1 concentration was observed from 10 to 1000 ng/lane. Thus, this represents a sensitive and specific method for quantitating small amounts of IL-1 in complex protein mixtures.