Copper Iodide Staining of Proteins on Solid Phases

Abstract

Copper iodide staining and silver enhancement are recent implementations of copper-based protein assays, and are designed to quantitate proteins adsorbed to solid surfaces, such as nitrocellulose, nylon, polyvinylenedifluoride, and polystyrene (1–5). The binding of cupric ions to the backbone of proteins under alkaline conditions and their consequent reduction to the cuprous state are the basis of popular assays of protein in solution including the biuret, Lowry, and bicinchoninic acid methods (see Chapters 2 and 3). In the case of copper iodide staining, it is thought that the protein binds copper iodide under highly alkaline conditions. Sensitivity, speed, reversibility, low cost, and lack of known interfering substances (including nucleic acid) are among the virtues of this protein assay (4–6). One interesting use of copper iodide staining is the quantification of protein adsorbed to microtiter plates (5). This information is particularly useful for quantitative ELISA and protein binding when radioactive measurements are inconvenient or undesirable (e.g., chemical modification affects the properties of the protein of interest). The precision of the determination of protein adsorbed to the microtiter plate by copper iodide staining is typically about 10–15%. The high sensitivity of copper iodide staining (about 50 pg/mm²) may be increased an additional 10-fold by a silver-enhancement procedure, allowing the detection of protein down to about 5 pg/mm² (6). Protein concentrations may be estimated from copper iodide staining from very dilute protein solutions or when only small amounts of a precious protein are available.