The western blot is a very useful and widely adopted lab technique, but its 
execution is challenging. The workflow is often characterized as a "black box" 
because an experimentalist does not know if it has been performed successfully 
until the last of several steps. Moreover, the quality of western blot data is 
sometimes challenged due to a lack of effective quality control tools in place 
throughout the western blotting process. Here we describe the V3 western 
workflow, which applies stain-free technology to address the major concerns 
associated with the traditional western blot protocol. This workflow allows 
researchers: 1) to run a gel in about 20-30 min; 2) to visualize sample 
separation quality within 5 min after the gel run; 3) to transfer proteins in 
3-10 min; 4) to verify transfer efficiency quantitatively; and most importantly 
5) to validate changes in the level of the protein of interest using total 
protein loading control. This novel approach eliminates the need of stripping 
and reprobing the blot for housekeeping proteins such as β-actin, β-tubulin, 
GAPDH, etc. The V3 stain-free workflow makes the western blot process faster, 
transparent, more quantitative and reliable.