Near-infrared fluorescence detection permits accurate imaging of loading controls for Western blot analysis

• Published in: Analytical biochemistry Vol. 375; no. 1; pp. 156 - 158
• Main Authors: Weldon, Sally, Ambroz, Kristi, Schutz-Geschwender, Amy, Olive, D. Michael
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.04.2008
• Subjects: Blotting, Western - methods; Blotting, Western - standards; Fluorescence; Humans; Jurkat Cells; Reference Standards; Spectroscopy, Near-Infrared - methods
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2007.11.035

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 β-actin did not reliably identify differences in sample loading, and extended antibody incubations caused a failure to discriminate differences in target protein levels. Here, β-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 R2 values as high as 0.9939. Longer antibody incubations continued to detect differences in protein level and load-dependent responses became more linear.