So this is my first time posting and I've got a bit of a riddle I'm trying to figure out, or at least make some sense of. I've run 3 blots, 1 as an antibody trial, and then 2 for actual data and while they both provide reasonable signal they have bands in different places. To make things more complex, a few conditions changed between the trial and the actual data blots but I can't figure out why I'm seeing the changes I'm seeing.
For more information: I'm using a ferritin light chain (FTL) antibody and it should produce a band of ~20 kDa. In my trial using samples of Hippocampus, Cortex, and Spleen (as a marker) I produced a nice band at 20 kDa with a prominent minus primary band at about 35 kDa in the brain samples (not the spleen). When I ran my other two blots they were solely Cortex samples and the same Spleen marker. They were originally tested for a different protein and I stripped and reprobed for FTL. Now the samples show a prominent band above 220 kDa and the Spleen (same sample) shows a weaker band at 20 kDa and a prominent band above 220 kDa. The minus primary is gone but I've observed this multiple times after stripping a blot.
So the question is: Why have the bands moved? I know FTL can exist natively in an aggregate of up to 450 kDa but don't know what would cause the aggregation. The blots ran fine and the data from the initial antibody matched historical data perfectly. I can't imagine that the reprobe protocol would cause a shift (I would understand weaker signal though).
Any help is appreciated.
Extra Info: Using Criterion XT 12% gels with XT buffer and reducing agent (uses TCEP). The membranes are nitrocellulose.