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 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 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.