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 order to have confidence in densitometric data from western. This is mainly due to the various steps, techniques, reagents, and detection methods that are used to obtain the associated data. In fact, the gel for the wild type was accidentally loaded with more of the sample. Chemiluminescent western blotting has been in common practice for over three decades, but its use as a quantitative method for measuring the relative expression of the target proteins is still debatable. An image of the membrane is then capturedeither on to film or using a digital imagerto allow for analysis. Western blotting begins with separating a mixture of proteins on a gel, transferring them to a membrane, and detecting one or more targets of interest with labeled antibodies. 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. Basic principles of western blot image analysis.
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