Open AccessEffects of light spectrum in flatbed scanner densitometry of stained polyacrylamide gels
Author(s) -
Han Tan,
Tuck Wah Ng,
Oi Wah Liew
Publication year - 2007
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/000112402
Subject(s) - densitometry , polyacrylamide , scanner , optical density , polyacrylamide gel electrophoresis , chemistry , optics , materials science , chromatography , microbiology and biotechnology , biology , physics , biochemistry , enzyme
Coomassie™ Brilliant Blue is arguably the most common dye used in staining polyacrylamide electrophoresis gels. A densitometric analysis into the extent of coloring by the dye can provide a quantitative measure of the amount of protein present. In this work, the experimentally determined spectral optical density distributions of Coomassie Blue-stained gels with various masses of proteins present allowed the optical density range response using different illumination to be predicted. Numerical modeling uncovered the spectrum of light has pronounced effects on the optical density range; wherein a higher scale translates to improved sensitivity. Generally, two factors contribute positively to this effect: (i) having the spectrum peak close to the 593-nm maximal absorption band of Coomassie Blue and (ii) possessing a spectrum width as narrow as possible. Based on this, we demonstrate that cost-effective densitometry can be achieved using a flatbed scanner with red light-emitting diode (LED) illumination, yielding results comparable to that of a commercial densitometer.