I have noticed Actin works well as a loading control in western blot when using a single cell line and whole cell lysates. However, when I make comparisons between cell lines then Actin does not look equal even, though the same amount of protein is loaded. Actin is a house keeping gene but I imagine Actin levels would vary between cell lines. How would this come into play with cells that are more spread out and have larger cytoplasm to nuclear ratio vs a more compact cell with a small cytoplasm? A cell type with larger cytoplasm would be enriched for cytoplasmic proteins. Not sure if any other loading control would overcome this issue.