I'm starting to run some chemiluminescent protein dot blots for a simple verification of tau pathology within our mouse model. We have validated that our AT8 antibody works using IF and DAB approaches, however I'm trying to discern hyperphosphorylated tau from tangles. The antibody we have for tangles shows a lot of non-specific labeling during IF since it is from a mouse host.

This dot blot was run on a nitrocellulose membrane. Membranes were dry during spotting, dried for an hour after spotting, blocked using 3% BSA in TBS-T for 30 minutes, incubated in a biotinylated primary overnight, incubated in an avidin-biotin-HRP conjugate overnight, then developed using chemiluminescent substrate.

The top row is the tauopathy model, and the bottom row is wildtype. Each dot is from a unique animal. The protein concentration of these spots was very high (>3000 ng/uL). Since it looks like the signal is localized to the center, I'm guessing that the protein didn't evenly distribute during dotting and depleted the chemi substrate. Would this reasonably lead to this pattern? How can I achieve a more even distribution of protein through my dot?