Quantifying the intensity of the bands on a Western blot can provide valuable information about protein expression levels. Image software allows for accurate and efficient quantification of Western blots by measuring the intensity of the bands. To begin the quantification process, the first step is to upload the image into the image software. Once the image is uploaded, it may appear with some imperfections, such as bands that are not aligned or a background that needs to be removed.Įditing the Image: Inverting and Rotating There are two ways to do this - either by going to "File" and selecting "Open" or simply dragging the image into the software. To correct any alignment issues with the bands, we can use the editing functions of the image software. First, we can invert the image by going to the "Edit" menu and selecting "Invert." This will ensure that the bands appear in a straight line. If the bands are still slightly tilted, we can use the "Transform" option in the "Image" menu to rotate the image by a specific degree. Experimenting with different rotation angles will help achieve the desired alignment. In addition to aligning the bands, it is often necessary to remove the background from the image to obtain clear curves for analysis. This can be done by adjusting the brightness and contrast of the image. Using the "Adjust" option in the "Image" menu, we can navigate to "Brightness and Contrast" and make adjustments until the background is no longer visible. It may require some trial and error to find the optimal settings. Once the image has been edited to perfection, we can proceed to select the bands of interest for analysis. This is done by using the selection tool, often represented by a rectangle icon. The selection should encompass the bands while avoiding unnecessary areas. It is crucial to ensure that the selection includes all the bands of interest and that there is enough room underneath for further analysis. Once the selection is made, we can proceed to the analysis phase. The next step in quantifying Western blots is calculating the ratios. This involves dividing the intensity values of the bands by a control value. In this case, the control value is derived from the housekeeping or actin bands. By dividing the intensity values of the bands of interest by the control value, we can obtain ratios that represent the relative expression levels. Normalizing the Protein of Interest to Housekeeping Proteins Calculation of ratios should be performed for both the housekeeping genes and the protein of interest in both the control and treatment groups. See the list of citable software and publications using Fiji for additional citation information.To normalize the protein of interest, we divide its ratio by the ratio of the housekeeping proteins. Rueden, C., Dietz, C., Horn, M., Schindelin, J., Northan, B., Berthold, M. (2016 Dec 7), " SCIFIO: an extensible framework to support scientific image formats.", BMC Bioinformatics 17(1): 521, PMID 27927161, doi: 10.1186/s1285-0, ( on Google Scholar). (2012), " A Graphical User Interface for R in a Rich Client Platform for Ecological Modeling.", Journal of Statistical Software 49(4): 1-19, ( on Google Scholar). (2010), " BoneJ: Free and extensible bone image analysis in ImageJ", Bone 47(6): 1076-1079, PMID 20817052, ( on Google Scholar).Īustenfeld, M. (2012), " NIH Image to ImageJ: 25 years of image analysis", Nature methods 9(7): 671-675, PMID 22930834, ( on Google Scholar). (2017), " ImageJ2: ImageJ for the next generation of scientific image data", BMC Bioinformatics 18:529, PMID 29187165, doi: 10.1186/s1285-z, ( on Google Scholar). In general, please cite these projects-their long-term future depends on it! If journal reference limits interfere, the plugin-specific publications should take precedence. The general paper of ImageJ distribution(s) used-e.g., if you used Fiji, cite the Fiji paper.Any specific publications of plugins used.For information on how ImageJ projects are funded, see Funding.For information on who maintains these projects, see Governance.For information on who contributes to these projects, see Contributors.This page describes how to cite SciJava projects.
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