Why is the actual western blot band size different from the predicted band size?

One reason is because modifications like phosphorylation or glycosylation can increase the size of proteins, affecting their migration in the gel. Another reason is because alternative splicing can produce proteins of different sizes from the same gene, leading to variability in protein products. Proteins may also have multiple isoforms of different sizes, which can vary depending on tissue and experimental conditions. Additionally, protein multimerization (such as dimerization) can affect band appearance, especially if interactions occur under specific conditions. Proteins also often begin as pro-proteins and undergo cleavage to reach their active form, changing their size in the process. Lastly, differences in amino acid composition (particularly charged vs. non-charged residues) can influence protein migration.