I am monitoring the expression of mitochondrial membrane-bound proteins in European pear peel tissue via quantitative fluorescent western blot analysis. I am normalizing on membrane with stain-free technology and have determined a linear dynamic range of protein detection (0.1ug-30ug).

I attempt to load equal amounts of total protein per lane (15ug). I have found the modified Lowry et al. Biorad Reductant and Detergent (RC DC) protein quantitation to be relatively imprecise, lending discernible differences in total protein per lane despite my attempts to load equal amounts. I use this method of total protein quantitation due to the relatively high amounts of detergent my tissue type and protein location requires.

Can I still use total protein normalization despite unequal lane to lane total protein load if I include a BSA control within my linear dynamic range (~15 ug) on every blot?

The goal is to compare protein of interest expression over many different chemical treatment combinations over time.

Thank you,

Evan