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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!

-Epigeneticist-

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?

-bob1-

Thanks!

-Epigeneticist-