Hi! I'm working on a protein whose size is 58kDa, I first wanted to test the antibody using 25ug iPSCs to check the concentration for the antibody that I need to use and the band would always be around 58kDa. (using 12% homemade acrylamide gel)

I then moved on to protein that's extracted from E15.5 embryonic heads, I ran it using 10% homemade acrylamide gel and used 25ug protein and compared it with the iPSCs. it showed some smearing but showed the primary band at the correct size.

Next, I wanted to use it on my samples, I protein that was extracted from E13.5 mouse embryonic bodies that have different genotypes of a mutation in this protein (WT, heterozygotes, and homozygotes). Therefore. I used the three genotypes and used their protein (20ug, since I figured the smear was from high protein concentrations) using a 10% gel but the band that I got was stuck on the top (around 250kDa)

So I repeated it again and used the E15.5 (20ug) as a control next to the E13.5 but I got the same result. What am I doing wrong and how can I fix it?

I'm adding pictures of all the results, the red square indicates the protein I'm interested in. In picture 3 I know the protein for the iPSCs was degraded, so just ignore it.

Also for context:

I extract using a pestle with RIPA buffer and proteinase inhibitor. I run the gel at 80V

Another thing that's weird that I noticed is that the B Actin (45kDa in the last picture) shows multiple bands in some sections, does anyone have a clue why?