Accurate step wedge calibration for densitometry of electrophoresis gels

Accurate step wedge calibration is important for reliable quantitative densitometry of electrophoresis gels. In this work, we outline the theory and verified by experiment, the effect that the nonlinear response of detectors at low irradiance levels has on accurate optical density (OD) calibration of step wedges. It was found that OD value departures from stated do exist and thus supports the case of calibration. Halogen broadband light was shown to be generally unsuited as light source for step wedge calibration even though applied at highest illumination before saturation, unless correction approaches which add complexity to the process are applied. Monochromatic light was determined best suited for use, provided that the irradiance level was high enough for the calibration to be conducted fully in the detector’s linear range.