Our lab has started working with the Amersham AI600 imager, which has given some very sensitive blots, thus reducing the amount of antibody we use. However, I notice that the autocorrect function gives a lot of background and band saturation, and this is usually seen even at lower exposure times (semi-auto frequently tells me I have to expose for greater than 10 minutes, but I know that is almost never true). This, I have been finding myself adjusting the "contrast" (if that is what it is rather than black intensity), which brings out the contrasts between bands better and reduces the saturation of the highly intense bands (the bands would remain the same intensity according to ImageJ, whereas everything else is reduced). I was justifying this as merely doing "shorter" exposures, albeit in reverse. However, I keep wondering whether this might be considered image manipulation. Comparing the ImageJ quantification between the finished picture and the original TIFF (which, BTW, is not the same as the original autocorrected image) is giving me nightmares. Does anyone have a good read on whether what I'm doing is acceptable, or should I just do my quantification on the original, non-autocorrected TIFF?
And here are the numbers I got:
BR (3.0 uM) 15997.095
Sig (3.0 uM) 7774.782
BR (3.0 uM) 5784.246
Sig (3.0 uM) 2644.447
BR (3.0 uM) 11837.125
Sig (3.0 uM) 2924.962
Edited by JDSBlueDevl, 12 August 2015 - 08:06 PM.