I have recently done my first western blot and I am doing data analysis to quantify my blot. I have labelled my membrane against inactive GSK3 and active GSK3 which are phosphoproteins so I am using total GSK3 as an internal loading control. I have read some guides and handbooks about western blot data analysis and I have seen that some of them calculate a lane normalisation factor to account for variations in signal intensities of the loading control. For all loading control bands in each lane, they divide by the loading control band with highest intensity to get the lane normalisation factor. Then for each band of the target protein of interest they divide by the lane normalisation factor to get the normalised intensity.
I was wondering in general with western blots is it good practice to calculate the lane normalisation factor when doing the data analysis? Any insights are appreciated.