Protein Detection Using Reversible Metal Chelate Stains

Electrophoretically separated proteins may be visualized using organic dyes, such as Ponceau red, Amido black, fast green, or most commonly Coomassie Brilliant Blue (
1
,
2
). Alternatively, sensitive detection methods have been devised using metal ions and colloids of gold, silver, copper, carbon, or iron (
3
–
12
). Metal chelates form a third class of stains, consisting of organometallic complexes that bind avidly to proteins resolved in polyacrylamide gels or immobilized on solid-phase supports (
13
). The metal chelate staining procedures are simple, requiring reagents that are easily prepared, are stable at room temperature, and can be reused several times without loss of sensitivity. The staining procedures are relatively inexpensive, since they do not utilize precious metals, such as gold or silver. Like Ponceau red stain, the metal chelate stains are readily reversible. Complexes form at acidic pH and elute on increasing the pH to 7.0–10.0. Metal chelates can be used to detect proteins on nitrocellulose, PVDF, and nylon membranes as well as in polyacrylamide gels. The metal complexes do not modify proteins, and are compatible with immunoblotting, lectin blotting, mass spectrometry, and Edman-based protein sequencing (
13
–
17
). Metal chelate stains are suitable for routine protein measurement in solid-phase assays owing to the quantitative stoichiometry of complex formation with proteins and peptides (
15
,
16
). Such solid-phase protein assays are more sensitive and resistant to chemical interference than their solution-based counterparts (
15
).