I have recently done a Western Blot and I am doing data analysis on my blots. I am studying the protein GSK3, which is a phosphoprotein.

I have labelled my membrane against active GSK3 and inactive GSK3. I have also labelled the membrane against total GSK3. I have used Ponceau S as a loading control. From my Ponceau S staining I have seen that there are variations in the amounts of protein loaded per lane. So there are variations in the amounts of total GSK3 per lane (the intensities of the bands are not all equal).

I am wondering if the band intensities of total GSK3 are not consistent among lanes, in this case would using total GSK3 be a good loading control to normalize my data on? I have read for the case of phosphoproteins, you can normalise to the total protein (using an antibody that recognises all forms of the protein of interest).

I have read that loading controls are endogenous proteins that are unaffected by experimental conditions and used as an indicator of sample concentration. But if the amounts to total GSK3 vary between the lanes, how can this be a good loading control? Similarly, why do many researchers use Ponceau S as a loading control, if the band intensities among lanes vary (if you don't load equal amounts of sample)?

In other words, for a protein to be a loading control, does it have to be in equal amounts for all your samples/lanes?

Any insights are appreciated.

  • $\begingroup$ Can you clarify what you are loading? Purified GSK3, total protein, or something else. $\endgroup$
    – Michael_A
    Commented Mar 23, 2021 at 14:11
  • $\begingroup$ I am loading 20 micrograms of brain lysates per lane (total protein that is present). $\endgroup$
    – ceno980
    Commented Mar 24, 2021 at 2:33

1 Answer 1


Controls do not need to loaded in equal amounts per say, but they do need to be expressed relatively equally between lanes. They need equal expression, such that controls can be used to normalize values of the experimental proteins.

It is common to use housekeeping genes, such as beta actin, which are usually expressed equally between treatments to control for loading. Important to ensure treatments don't affect those proteins.

For examples and rationale, see:





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