Hi! I used the NEB PNGase-F denaturing protocol to denature some lysates and ran them on Western. I usually load 20µg of protein on western; so I did that with my un-denatured lysate control as well as my lysates, using the same amount of the lysate/denatured lysate in the sample prep

2µL lysate/denatured lysate

2µL DTT

3µL lammeli buffer sds (6x)

8µL MilliQ water

Samples were then briefly vortexed, spun, and boiled at 95C for 5 minutes before being run on a gel and probed for the protein. The denatured protein samples show the correct band I want, but when using GAPDH as my loading control, it is evident that the same amount of protein was not loaded. I cannot find anything on the internet about using different amounts of protein for loading after this...

I also made a silly mistake after and thought I could BCA my denatured samples to get the exact concentration of them, but all the glycerol interfered with the BCA and gave super high protein results.