Pyrogallol red-molybdate: a reversible, metal chelate stain for detection of proteins immobilized on membrane supports.
Certain metal complexes selectively interact with proteins immobilized on solid-phase membrane supports to form brightly colored products. The metal chelates form protein-dye complexes in the presence of metal ions at acidic pH but are eluted from the proteins by immersing membranes in a solution of basic pH that contains other chelating agents. The reversible nature of the protein staining procedure allows for subsequent biochemical analyses, such as immunoblotting, N-terminal and internal protein sequencing. Among the metal complexes evaluated to date, the triazine dye-ferrous complexes (ferene S, ferrozine) and the ferrocyanide-ferric complexes provide the most sensitive detection of proteins immobilized on membranes. While the pyrogallol red-molybdate complex is commonly used in solution-based total protein assays, its utility as a reversible stain for proteins immobilized on membranes has not been reported. Pyrogallol red-molybdate complexes readily stain proteins on nitrocellulose and polyvinyl difluoride membranes with similar sensitivity as ferrozine-ferrous complexes. Analysis of charge-fractionated carrier ampholytes and synthetic polymers of different L-amino acids indicate that binding is prominently via protonated alpha and epsilon-amino side chains. Carbamylation of amino groups in bovine serum albumin substantially diminishes pyrogallol red-molybdate binding to the protein. The stain is reversible, resistant to chemical interference, and compatible with immunoblotting.
Shojaee
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Microvascular Research Laboratory, Boston University, MA 02215, USA.
Patton
W F
WF
Lim
M J
MJ
Shepro
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D
D002614:Chelating Agents / Q000737:chemistry* / Q000378:metabolism; D003101:Collodion / Q000737:chemistry* / Q000378:metabolism; D004396:Coloring Agents / Q000737:chemistry* / Q000378:metabolism; D015151:Immunoblotting; D008567:Membranes, Artificial; D015394:Molecular Structure; D008982:Molybdenum / Q000737:chemistry* / Q000378:metabolism; D011145:Polyvinyls / Q000737:chemistry* / Q000378:metabolism; D011748:Pyrogallol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000032:analysis*; D005465:Fluorocarbon Polymers
D002614:Chelating Agents; D003101:Collodion; D004396:Coloring Agents; D008567:Membranes, Artificial; D008982:Molybdenum; D011145:Polyvinyls; D011748:Pyrogallol; D012710:Serum Albumin, Bovine; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C058731:pyrogallol sulfonphthalein; C024687:sodium molybdate(VI)