The script will overwrite the initial excel file, saving all the data in a sheet named “DATA,” the filtered data between exposure times in a sheet named “DATA_FILTERED,” and the coefficients of the linear model in a sheet named “MODEL.” Comparison of the slopes obtained provides a more accurate representation of the relative amounts of proteins in different samples than would be achieved by comparing individual images.Įxport the analysis results. The advantage of our procedure is that the signals are quantified across an interval of different exposures within the linear range for all the samples, where the differences between them are constant. Different images with different exposure times were utilized to generate the linear range for each protein to be quantified. Here, we describe an improved, simple protocol for western blot quantification taking advantage of chemiluminescent detection by ccd cameras and ImageJ and R software. Therefore, it is urgent to establish a simple, reliable method to quantify western blot signals to draw accurate conclusions in the studies. Fluorescence-based techniques using fluorophore-conjugated secondary antibodies are not commonly employed in most laboratories but provide a wider range of linear correlation between signal and protein abundance ( Huang et al., 2019). Previous articles quantifying chemiluminescence western blots have relied on the use of single images of signals from different samples that are sometimes saturated, leading to incorrect outcomes. Usually, these are housekeeping proteins including actin, tubulin, and glyceraldehyde 3-phosphate dehydrogenase. To assess accurate quantification, it is required to perform data normalization using proteins that do not vary due to experimental conditions. In addition, reviewers and editors are eager to have trusty quantitative data regarding western blot analyses ( Murphy and Lamb, 2013 Taylor et al., 2013 Janes, 2015 McDonough et al., 2015 Bass et al., 2017 Pillai-Kastoori et al., 2020). However, a proper quantification to determine the amount or the corresponding protein modification detected is still missing in most of the articles. Since 2000, when 7,300 papers were published, there has been a constant increase in the number of articles reporting the use of this method ( Moritz, 2020), reaching almost 22,600 in 2021. Since the development of western blotting in 1979 ( Towbin, H, PNAS 1979), more than 382,000 articles have been published using this technique ( Western blot, PubMed, October 11, 2022). Western blot is the most widely used and accepted method to detect proteins of interest using specific antibodies. Proteins are key biomolecules for the correct functioning of living animals therefore, it is required not only to detect but also to accurately quantify them.
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