METAL-CHELATES AS REVERSIBLE STAINS FOR DETECTION OF ELECTROBLOTTED PROTEINS - APPLICATION TO PROTEIN MICROSEQUENCING AND IMMUNOBLOTTING

Coomassie brilliant blue and Ponceau red have traditionally been used to stain electroblotted proteins, since they are compatible with exist ing N-terminal and internal protein microsequencing as well as with im munoblotting procedures. With recent improvements in sequencing and im munoblotting technology, detection of significantly smaller amounts of protein has become necessary. Metal complexes were evaluated as alter natives to conventional stains. Electroblotted proteins were detected by blocking nonspecific sites with polyvinylpyrrolidone-40 followed by incubation in metal chelate solutions at acidic pH values. Two of the most promising metal chelate stains were the Ferrozine/ ferrous compl ex and the ferrocyanide/ferric complex. Both stained a wide variety of proteins and peptides quantitatively. Dot blots and 1D and 2D electro blots were successfully stained using iron chelates. When these two st ains were utilized in combination, they were of equivalent sensitivity to colloidal gold stain. The reversibility of the metal chelate stain s was substantiated by incubating stained membranes at neutral to basi c pH in the presence of 20 mM ethylenediaminetetraacetic acid to rapid ly elute the complexes from the bound proteins. The chelate stains wer e determined to be fully compatible with immunoblotting, N-terminal, a nd in situ internal protein microsequencing. (C) 1994 Academic Press, Inc.