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 existing N-terminal and internal protein microsequencing as well as with immunoblotting procedures. With recent improvements in sequencing and immunoblotting technology, detection of significantly smaller amounts of protein has become necessary. Metal complexes were evaluated as alternatives 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 complex and the ferrocyanide/ferric complex. Both stained a wide variety of proteins and peptides quantitatively. Dot blots and 1D and 2D electroblots were successfully stained using iron chelates. When these two stains were utilized in combination, they were of equivalent sensitivity to colloidal gold stain. The reversibility of the metal chelate stains was substantiated by incubating stained membranes at neutral to basic pH in the presence of 20 mM ethylenediaminetetraacetic acid to rapidly elute the complexes from the bound proteins. The chelate stains were determined to be fully compatible with immunoblotting, N-terminal, and in situ internal protein microsequencing.
Patton
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Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
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D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002614:Chelating Agents; D004396:Coloring Agents; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006454:Hemoglobins / Q000032:analysis / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting / Q000379:methods*; D008670:Metals; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000737:chemistry; D013194:Staining and Labeling
D000596:Amino Acids; D002614:Chelating Agents; D004396:Coloring Agents; D006454:Hemoglobins; D008670:Metals; D011506:Proteins; D012710:Serum Albumin, Bovine