Background-Free Protein Detection in Polyacrylamide Gels and on Electroblots Using Transition Metal Chelate Stains

Abstract

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 devi sed using metal ions and colloids of gold, silver, copper, carbon, or iron (3–12). Metal chelates form a third class of stains, consisting of transition metal complexes that bind avidly to proteins resolved in polyacrylamide gels or immobilized on solid-phase membrane supports (13–27). In recent years, metal chelate stains have been designed and optimized specifically for compatibility with commonly used microchemical characterization procedures employed in proteomics. The metal chelate stains are simple to implement, and do not contain extraneous chemicals such as glutaraldehyde, formaldehyde, or Tween-20 that are well known to interfere with many downstream protein characterization procedures.

Metal chelates can be used to detect proteins on nitrocellulose, poly(vinylidene difluoride) (PVDF), and nylon membranes as well as in polyacrylamide and agarose gels. The metal complexes do not modify proteins, and are compatible with immunoblotting, lectin blotting, mass spec-trometry, and Edman-based protein sequencing (13–17,22–27). 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).