Effects of light spectrum in flatbed scanner densitometry of stained polyacrylamide gels.
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.
Tan
Han Yen
HY
National University of Singapore.
Ng
Tuck Wah
TW
Liew
Oi Wah
OW
D003720:Densitometry / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D008027:Light; D013057:Spectrum Analysis