It is probably one of the most recurrent questions when it comes to WB.
Some argue that using GAPDH or other proteins as normalization (HKP) factors is the way. Nevertheless, validation is almost never required/shown.
Others advocate the use of total protein (lane - TPN) normalization which is faster, but too dependent on the exposure time and image quality.
How do you tackle the problem in your lab?
Total protein lane; we use Ponceu staining for this. Obviously with the assumption that the image quality is as good (or bad) everywhere in the blot....
I have been taught to do the same with silver staining/coomassie back. Now we have a stain-free TGX gel system from BioRad, but the data is somehow incoherent at times (plus bending lanes complicate normalization).
But IMO still better than GAPDH approximation
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