Have you found any related forum topics? If so, cross-link them.But since it is difficult to place the line on the bell-shaped curve exactly at the same spot it’s hard to tell if it overlapping boxes affect the quantified band intensities. Quantifications of Western Blots with ImageJ by Hossein Davarinejad This protocol will allow you to relatively (no absolute values) quantify protein bands from. Inexpensive computer imaging technology was used to assess levels of insulin-like growth factor-I (IGF-I) on dot blots (DB) and alpha-Actinin on Western blots (WB). draw around the lane you wish to quantify 3. I quantified the bands separately and with overlapping boxes. The method is also preferred when the bands you need to quantify are very faint. Below is an example of a Western Blot with six lanes showing the. I don’t know if the overlapping of boxes of two bands affects the quantified values of each band The protocol is based on the excellent instructions for using ImageJ to quantify bands. Would this affect quantification considering that the white space around the two bands is overlapping but the overlapping boxes don’t reach over the actual band? You can rotate your image if needed using Image > Rotate. Citation: Kondo Y, Higa S, Iwasaki T, Matsumoto T, Maehara K. Your lanes must be horizontal in order for Image J to analyze them properly. In this method, filters and fluorescent dyes are optimized to enhance detection sensitivity to a level similar to that of the chemiluminescence method. I want to load 50 ug/20 ul/well of SDS-PAGE for Western blot and I have protein concentrations 6.18, 4.9, 5.76, 6.53, 4.43, 5.83, 5.11& 7.46 ug/ul. Plots of CHEMI_10242021_154542_fis_br_se_cropped_FIS_marked_bell curves.tif (523.5 KB)Ĭan the boxes from different bands overlap? I need a certain box size to fit the largest band, but this leads to the box around one band overlapping in the white space of the box of the next band (not over the next band, only the white space). Here, we describe a method to detect fluorescence in western blots using fluorescence microscopy to obtain high-resolution images. *Use the wand and get a quantification value from the intensity of my band.ĬHEMI_10242021_154542_fis_br_se_cropped_FIS_marked.tif (65.6 KB) Due to the technical knowledge required for densitometry analysis together with the resources availability, standard office scanners are often used for the imaging acquisition of developed Western blot films. From the generated plots I mark the bell shaped curve with the line tool Western blot quantification method constitutes a critical step in order to obtain accurate and reproducible results.I have a question regarding western blot analysis using imageJ.
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