Fold-changes different when quantifying same protein via IF and Western - (Jun/19/2012 )

Hi,

I'm using IF and western blot to quantify the fold change of fibronectin in cells with and without treatment. The fold changes are much lower when I quantify my IF pics versus when I quantify the protein via western blot. For example, with one treatment my western shows a 6.7-fold increase, while my IF only shows a 1.7-fold increase. It seems like the IF fold-changes plateau more quickly, never going above a 2.5-fold increase. I'm using the same antibody, and it's good for both applications.

Does anyone know why my IF and western quantifications wouldn't be similar, or even proportional? (My PI thinks they should be.)

First off, IF isn't really a quantitative technique, and westerns are only just if you can control all the outside factors, so I am not at all surprised to see that you find differences.

If al things went well, you could perhaps expect to see the same fold-change between IF and western, but you would have to control for the sensitivity and specificty of each assay and compare how each technique works to tell you if you should expect similar values.