A Method for Greater Reliability in Western Blot Loading Controls: Stain-Free Total Protein Quantitation

This application note compares the linearity of a series of dilutions measured by stain-free total protein measurement as well as immunodetection of three housekeeping proteins (ß-actin, ß-tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found that immunodetection-based measurements of ß-actin, ß-tubulin, and GAPDH protein levels neither showed good linearity nor accurately indicated 10–50 μg of HeLa cell lysate loading levels. By contrast, stain-free total protein measurements exhibited great linearity in the same loading range and its linearity correlated with the immunodetection of a selection of low abundant protein targets: MEK, Akt, and Erk. This study demonstrated that stain-free total protein measurement serves as a more reliable loading control than housekeeping proteins, particularly in the loading range commonly used for cell lysates, 10–50 μg.