Western blot analysis is a process where one quantitates the amount of signal detected on a western blot from a specific band. Within the software boxes are drawn around bands of interest and values are displayed. The values obtained can be used to compare untreated to treated experimental samples.
Due to the fact there can be variability in loading and transfer efficiency from well to well, some method of normalisation should be used. There are two main ways to normalise values and that is by using an internal reference or housekeeping protein or by using a total protein stain to normalise to total amount of sample loaded per well. In either case this allows one to see if there is significant differences between samples. This is usually done within the software that the western blot was imaged with or in third party freeware analysis software.
With improvements in instrumentation some freeware analysis programs cannot cope with the dynamic range of the newer instruments like the LI-COR Odyssey CLX and FC systems therefore it is recommended when using LI-COR instruments to use the Image Studio software for performing data analysis to ensure one can analyse the entire dynamic range of the data file.