Intrinsic protein fluorescence interferes with detection of tear glycoproteins in SDS-polyacrylamide gels using extrinsic fluorescent dyes.
Intrinsic protein fluorescence may interfere with the visualization of proteins after SDS-polyacrylamide electrophoresis. In an attempt to analyze tear glycoproteins in gels, we ran tear samples and stained the proteins with a glycoprotein-specific fluorescent dye. The fluorescence detected was not limited to glycoproteins. There was strong intrinsic fluorescence of proteins normally found in tears after soaking the gels in 40% methanol plus 1-10% acetic acid and, to a lesser extent, in methanol or acetic acid alone. Nanograms of proteins gave visible native fluorescence and interfere with extrinsic fluorescent dye detection. Poly-L-lysine, which does not contain intrinsically fluorescent amino acids, did not fluoresce.
Zhao
Zhenjun
Z
The institute for Eye Research, North Wing, Rupert Myers Building, Gate 14, Barker st, UNSW Sydney, NSW 2052, Australia. z.zhao@ier.org.au
Aliwarga
Yulina
Y
Willcox
Mark D P
MD
D000180:Acrylic Resins / Q000009:adverse effects; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000032:analysis*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D006023:Glycoproteins / Q000032:analysis*; D006801:Humans; D012967:Sodium Dodecyl Sulfate; D013666:Tears / Q000737:chemistry*
D000180:Acrylic Resins; D005136:Eye Proteins; D005456:Fluorescent Dyes; D006023:Glycoproteins; C016680:polyacrylamide gels; D012967:Sodium Dodecyl Sulfate