Western blots are used to specifically measure the relative quantities of 
proteins of interest in complex biological samples. Quantitative measurements 
can be subject to error due to process inconsistencies such as uneven protein 
transfer to the membrane. These non-sample-related variations need to be 
compensated for by an approach known as normalization. Two approaches to data 
normalization are commonly employed: housekeeping protein (HKP) normalization 
and total protein normalization (TPN). In this study, we evaluated the 
performance of Stain-Free technology as a novel TPN tool for Western blotting 
experiments in comparison with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 
as a representative of the HKP normalization strategy. The target protein (TP) 
used for this study was MCM7, a DNA licensing replication factor, which was 
shown previously to be down-regulated by 20% in irradiated lymphoblastoid cell 
lines (LCLs). We studied the regulation of MCM7 with a multiplex Western 
blotting approach based on fluorescently labeled secondary antibodies and found 
that Stain-Free technology appears to be more reliable, more robust, and more 
sensitive to small effects of protein regulation when compared with HKP 
normalization with GAPDH. Stain-Free technology offers the additional advantages 
of providing checkpoints throughout the Western blotting process by allowing 
rapid visualization of gel separation and protein transfer.