Best western loading control for comparing different cell lines - (Jun/08/2014 )

Hi, 

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. 

Thanks!

You are correct, actin does vary between cell lines, and this is the same for  almost any other protein you care to name.  However, you could try a panel of different loading controls and fine one that is the least variable - some of the nuclear markers may be a better bet, perhaps histoneH3 or nucleolin?

Thanks!