title: Near-infrared fluorescence detection permits accurate imaging of loading controls for Western blot analysis.
abstract: Housekeeping proteins are typically chosen as internal loading controls for Western blot analysis because of their high, relatively constant expression. It was previously reported that antibodies against beta-actin did not reliably identify differences in sample loading, and extended antibody incubations caused a failure to discriminate differences in target protein levels. Here, beta-actin and GAPDH were evaluated as loading controls using near-infrared fluorescence. A load-dependent response in signal intensity was observed over a 250-fold range of sample concentrations, with R(2) values as high as 0.9939. Longer antibody incubations continued to detect differences in protein level and load-dependent responses became more linear.
authors.affiliation: LI-COR Biosciences, Lincoln, NE 68504, USA.
authors.forename: Sally
authors.initials: S
authors.lastname: Weldon
authors.forename: Kristi
authors.initials: K
authors.lastname: Ambroz
authors.forename: Amy
authors.initials: A
authors.lastname: Schutz-Geschwender
authors.forename: D Michael
authors.initials: DM
authors.lastname: Olive
mesh_terms: D015153:Blotting, Western / Q000379:methods* / Q000592:standards*; D005453:Fluorescence; D006801:Humans; D019169:Jurkat Cells; D012015:Reference Standards; D019265:Spectroscopy, Near-Infrared / Q000379:methods*