Misleading Westerns: Common Quantification Mistakes in Western Blot Densitometry and Proposed Corrective Measures

Figure 1

Detection of commonly used loading control protein SMA. Pooled pregnant human myometrial tissue homogenates extracted in 2D lysis buffer were run in 2-fold serial dilutions from 40 μg to 40 ng of total protein lysate per lane. (a) Representative Western blot images of membranes detected with chemiluminescence (black bands) or infrared fluorescence (red bands). O.D. data were calculated from band area and background-subtracted intensity for (b) nitrocellulose membranes detected by chemiluminescence, (c) nitrocellulose membranes detected by infrared fluorescence, (d) PVDF membranes detected by chemiluminescence, and (e) PVDF membranes detected by infrared fluorescence. The panels on the right side of subfigures (b-e) show the same O.D. data as each panel on the left but have been restricted to data obtained between 1.25 μg and 40 ng of total protein lysate per lane to magnify the display of these data. The area that has been magnified in the left panels are shown by red rectangles. In these magnified regions, linear regression models are fitted to the data as shown by straight lines. 3 independently prepared membranes were analysed with each detection method.