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Figure 2 shows a stylised western blot of increasing concentrations of protein, and the “signal intensity” as measured by a commonly used software-in this example the last five concentrations gave the same intensity measurement despite representing very different amounts of protein.
![quantification western blot imagej quantification western blot imagej](https://os.bio-protocol.org/attached/image/20180925/20180925025036_4173.jpg)
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. This video will help you to quantify your protein bands from western blot. This protocol offers an Image J macro/plugin that enable easy quantification of bands on western blots, dot blots, and fluorescent gels etc. Each pixel in a blot image has an x and y coordinate, in addition to an intensity value, which occupies the third dimension. A digital image of a blot can be thought of as data in three dimensions. Search 216,022,918 papers from all fields of science. A western blot image is made up of pixels, which contain information about how much signal was collected at each location in the image. Skip to search form Skip to main content Skip to account menu. Steps to assure quantitative data from western blots. The chemiluminescent film was saturated, so the higher level of tubulin in the wild type was not reflected when the intensity measurements were taken: actually when the same amounts of sample were loaded, there was no change in expression of Protein X in the two conditions. Semantic Scholar extracted view of 'A new background subtraction method for Western blot densitometry band quantification through image analysis software.' by G. In fact, the gel for the wild type was accidentally loaded with more of the sample. However, although the two tubulin controls look the same-and give the same intensity measurements using a simple image analysis tool-they do not represent the same underlying expression.