Note that saturation can be detected at higher exposures of the TrkA blot in the pseudo color images (red color). A rectangle of another region without signal was utilized as background (not shown in the images). The colored rectangles in both panels represent the membrane areas used for quantification. Images with different exposures showing pTrkA ( panel A) and TrkA ( panel B) signals were quantified in cultured sensory neurons after NGF stimulation for different stimulation times. Graphical overview.Īnalysis Data normalization Linear regression model Protein Quantification Western blot.Ĭopyright © 2023 The Authors exclusive licensee Bio-protocol LLC. This approach allows to quantify and compare protein levels from different conditions in a simple and reproducible way. The result is a linear regression model in which we use the slope of the signal increase within the combined linear range of detection to compare between samples. Images were processed with ImageJ and subsequently compared using R software. Here, we have developed a procedure based on the increase in chemiluminescent signal to obtain a representative value for each band to be quantified. However, there is no clear and common procedure to quantify the results obtained, resulting in variations due to the different software and protocols used in each laboratory. Western blotting is a universally used technique to identify specific proteins from a heterogeneous and complex mixture.
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