A method for detecting proteins immobilized on nitrocellulose membranes by in situ derivatization with fluorescein isothiocyanate.
A method for the fluorescent staining of proteins on nitrocellulose filters is described. The single step procedure uses a 100 microgram/ml solution of fluorescein isothiocyanate in sodium carbonate buffer, pH 9.5. The proteins are visible under uv light within 30 s and the staining reaction is virtually complete after 10 min. The method can detect a minimum of 50 ng protein/band providing a sensitivity similar to that obtained with anionic dye stains. The method is suitable for blots prepared from both isoelectric focusing gels and sodium dodecyl sulfate-polyacrylamide gels. The fluorescently labeled proteins can be probed using immunochemical techniques with the retention of fluorescence. The method can therefore be used to accurately locate antigens among a number of proteins and allows the sensitive and rapid detection of marker proteins directly on the blot.
Houston
B
B
AFRC Institute of Animal Physiology and Genetics Research, Edinburgh Research Station, Midlothian, Scotland.
Peddie
D
D
D015153:Blotting, Western / Q000379:methods; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007525:Isoelectric Focusing / Q000379:methods; D011506:Proteins / Q000032:analysis*; D013861:Thiocyanates
D005452:Fluoresceins; D005456:Fluorescent Dyes; D011506:Proteins; D013861:Thiocyanates; D003101:Collodion; D016650:Fluorescein-5-isothiocyanate