Jpd. Goldring et L. Ravaioli, SOLUBILIZATION OF PROTEIN-DYE COMPLEXES ON NITROCELLULOSE TO QUANTIFYPROTEINS SPECTROPHOTOMETRICALLY, Analytical biochemistry, 242(2), 1996, pp. 197-201
Proteins absorbed directly onto nitrocellulose membranes were stained
with amido black, ponceau S, colloidal silver, or Coomassie blue and s
olubilized in dimethyl sulfoxide and the absorbance was measured spect
rophotometrically. The optimal wavelength of each dye/protein/nitrocel
lulose solution was found to be at 625, 529, 420, and 600 nm, respecti
vely. A linear relationship was found between the protein concentratio
n and absorbance at the appropriate wavelength for all the stains with
individual purified proteins or protein mixtures. Protein (0.2-0.8 mu
g) can be determined with the colloidal silver and 2-30 mu g with the
other stains. Coomassie blue produced variable background staining of
the nitrocellulose and is therefore not recommended. Proteins transfe
rred electrophoretically to nitrocellulose from a sodium dodecyl sulfa
te-polyacrylamide gel were also stained with the above dyes and solubi
lized in dimethyl sulfoxide. Amido black was the most sensitive stain,
detecting proteins in the range of 1-10 mu g. Components of the gel i
nterfered with silver staining. (C) 1996 Academic Press, Inc.