![]() ![]() Panels A and B correspond to experiment c in panel C. Each colored line represents the results obtained in a single experiment (n = 3). Normalization was performed using the 5 min time point of NGF stimulation. ![]() ( C) Quantification of TrkA activation (pTrkA/TrkA ratio). Note that the saturation in the pseudo color images matches the flattering of the curves in the plots. The range between dashed lines indicates the exposures taken to optimize the linear regression model and to perform the quantification within linearity. On the right side of each panel there is a graph showing the signal increase curve for each time point. 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. Figure 2 shows a stylised western blot of increasing concentrations of protein, and the signal intensity as measured by a. This approach allows to quantify and compare protein levels from different conditions in a simple and reproducible way. This represents a general problem of quantifying western blots with simple image analysis software, which may be unable to discriminate between similar-looking bands that have fallen off the end of the linear scale. 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. Same tools can be used for DNA gel images, thin layer chromatography, Southern blot. ![]() Here, we have developed a procedure based on the increase in chemiluminescent signal to obtain a representative value for each band to be quantified. This demonstrates how to create a figure to display western blot results. Hi, can anyone please let me know how to quantify the total protein after performing western blot by ImageJ software and then how to normalize my protein of interest. 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. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |