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. 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 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. In fact, the gel for the wild type was accidentally loaded with more of the sample. Muscle Biology Laboratory, Department of Animal Sciences, Washington State University, Pullman, WA 99164-6310, USA. It also produces publishable figures which indicate exactly the quantification areas which is extremely important for a transparent measurement.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. Evaluating dot and Western blots using image analysis and pixel quantification of electronic images. I will try to get in contact with the authors and see if there might be a possibility to link it as plugin into ImageJ (e.g. It is freely available but not completely open source with access to the source code.
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