Copper Iodide Staining of Proteins and Its Silver Enhancement

  • Douglas D. Root
  • Kuan Wang
Part of the Springer Protocols Handbooks book series (SPH)


Copper iodide staining and silver-enhancement is designed to quantify proteins adsorbed to solid surfaces such as nitrocellulose, nylon, polyvinylidene difluoride (PVDF), silica, cellulose, and polystyrene (1-5) and has important applications in Western blotting and thin layer chromatography (3,6). The binding of cupric ions to the backbone of proteins under alkaline conditions and their reduction to the cuprous state is the basis of several protein assays in solution including the biuret, Lowry, and bicinchoninic acid methods (1, 2, 3,7 and see  Chapters 2 4). In the case of copper iodide staining, the protein binds copper iodide under highly alkaline conditions. This protein assay demonstrates sensitivity, speed, reversibility, low cost, and the lack of known interfering substances (including nucleic acid; refs. 4,5). Copper iodide staining is sufficiently sensitive to permit the quantification of proteins adsorbed to microtiter plates (5). The information is particularly useful for the quantitative interpretation of enzymelinked immunosorbent assay (ELISA) and protein binding experiments. 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 40 pg/μL) may be increased several fold by a silver-enhancement procedure that allows the detection of protein down to about 10 pg/μL, which is more sensitive than common solutionbased assays (7). The sensitivity of the assay can be increased by repeated applications of the protein on a membrane to concentrate it. 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 such as for the analysis of chromatography fractions.


Microtiter Plate Sodium Tartrate Flatbed Scanner Potassium Sodium Tartrate Potassium Tartrate 
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Copyright information

© Humana Press Inc., Totowa, NJ 2002

Authors and Affiliations

  • Douglas D. Root
    • 1
  • Kuan Wang
    • 1
  1. 1.Department of Biological SciencesUniversity of North TexasDenton

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