Total protein staining with Congo red as an alternative loading control for western blot analysis

For western blot analysis, a housekeeping protein, such as β-actin or glyceraldehyde-3-phosphate dehydrogenase, is used as loading control with the assumption that these proteins are stable. In practice, these internal loading control proteins vary with different cell states and tissue types. These internal standards are not appropriate for use with serum, extracellular secretion, cerebrospinal fluid analysis or for protein purification. We investigated total protein measurement using Congo red staining and found it to be a superior alternative to routine loading controls. Advantages include lower cost, technical simplicity and improved linear regression. We propose using Congo red staining for total protein immunoblotting to evaluate protein loading in western blots.