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Background – Having an adequate loading control for a western blot is essential for the interpretation of the results. There are two common loading control methods for western blots of proteins from plant material: (i) using specific antibodies to detect for a reference protein, such as actin, tubulin, or GAPDH (Li et al. 2011); and (ii) treating the membrane with Ponceau or Coomassie stains to assay the levels of a constitutively expressed protein, such as Rubisco (Zhang et al. 2017; Lim et al. 2018; Zhuo et al. 2014). Comparative studies in the mammalian biology field have determined that these loading control methods—antibody detection versus staining—are roughly equivalent in their linearity (Romero-Calvo et al. 2010; Wilender and Ekblad, 2011), and thus serve as comparable quality controls. In the plant biology field, it is sometimes debated as to whether staining for Rubisco is an appropriate loading control, due to the high abundance of this protein in the cell.
Results – We undertook an experiment to determine whether the range of detection of staining for Rubisco is similar to that of antibody-based detection of a reference protein. We loaded total protein extract from Nicotiana benthamiana leaves transiently expressing GFP into a gel at a range of effective sample volumes, and the resulting western blot was treated with anti-GFP antibodies as well as stained with Coomassie Brilliant Blue (CBB) (Fig. 1a). Quantification of the GFP bands in the western blot and the Rubisco bands in the CBB stained membrane indicated that these detection methods have similar linear correlations between the loading volumes of total protein extract and the detectable band intensities (Fig. 1b). In addition, quantification of a random protein of lower abundance in the CBB stained membrane also showed similar linearity (Fig. 1b).
Conclusions – These results indicate that CBB staining for Rubisco can be an appropriate loading control for western blots from plant material. This representative experiment is consistent with results from other western blot experiments that we routinely perform in our laboratory.