3 independently prepared membranes were analysed with each detection method. In these magnified regions, linear regression models are fitted to the data as shown by straight lines. The area that has been magnified in the left panels are shown by red rectangles. 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 panels on the right side of subfigures (b-e) show the same 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. (a) Representative Western blot images of membranes detected with chemiluminescence (black bands) or infrared fluorescence (red bands). 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. These findings apply to all Western blot studies, and we highlight quality control checks that should be performed to make Western blot data more quantitative.ĭetection of commonly used loading control protein α SMA. Using spiked proteins in a way that allowed us to control the total protein amount per lane, while only changing the amount of spiked proteins, we confirm that nonlinearity and saturation of densitometry data, and errors introduced from normalisation processes, can occur in routine assays that compare equal amounts of lysate. We also confirm that when data to be normalised are not directly proportional to protein abundance, it is a mistake to use the normalisation technique of dividing densitometry data from the protein-of-interest with densitometry data from loading control protein(s), as this can cause the normalised data to be unusable for making comparisons. We confirm that ghosting artefacts associated with overabundance of proteins of interest in Western blots can skew findings. Nonlinear densitometry data were observed when Western blots were detected using infrared fluorescence or chemiluminescence, and under different SDS-PAGE conditions. While ideal densitometry data are directly proportional to protein abundance, our data confirm that densitometry data often deviate from this ideal, in which case they can fit nonproportional linear or hyperbolic mathematical models and can reach saturation. Proteins analysed included αSMA, HSP27, ERK1/2, and GAPDH. We ran dilution series of protein lysates to explore the linearity of densitometry data. We assessed the reliability of Western blot data obtained to study human myometrial tissue proteins. These violations can lead to erroneous interpretations of data and may contribute to poor reproducibility of research. In the last decade, it has become apparent that assumptions underpinning these comparisons are often violated in studies reporting Western blot data in the literature. Densitometry data generated for Western blots are commonly used to compare protein abundance between samples.
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