title: Total protein or high-abundance protein: Which offers the best loading control for Western blotting?
abstract: Western blotting routinely involves a control for variability in the amount of protein across immunoblot lanes. Normalizing a target signal to one found for an abundantly expressed protein is widely regarded as a reliable loading control; however, this approach is being increasingly questioned. As a result, we compared blotting for two high-abundance proteins (actin and glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) and two total protein membrane staining methods (Ponceau and Coomassie Brilliant Blue) to determine the best control for loading variability. We found that Ponceau staining optimally balanced accuracy and precision, and we suggest that this approach be considered as an alternative to normalizing with a high-abundance protein.
authors.affiliation: Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1.
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Thacker
authors.affiliation: School of Public Health and Health Systems, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1.
authors.forename: Derrick H
authors.initials: DH
authors.lastname: Yeung
authors.affiliation: Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1.
authors.forename: W Richard
authors.initials: WR
authors.lastname: Staines
authors.affiliation: Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1; School of Public Health and Health Systems, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1. Electronic address: jgmielke@uwaterloo.ca.
authors.forename: John G
authors.initials: JG
authors.lastname: Mielke
mesh_terms: D015153:Blotting, Western / Q000379:methods*; D011506:Proteins / Q000032:analysis*
chemical_list: D011506:Proteins
keywords: Actin; Coomassie brilliant blue; Glyceraldehyde-3-phosphate dehydrogenase; Immunoblotting; Ponceau S