![]() 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. Blotting was performed using a mixture of rabbit- and mouse-derived primary antibodies to ADH and. 2 was imaged with the ChemiDoc MP (a) and then with film (b). ![]() The chemiluminescent western blot of the two-fold dilution series of the HeLa lysate with spiked-in ADH protein from Fig. 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. Defining the linear dynamic range of western blot detection. Four Simple Steps for Western Blot Quantification 1. So on to the four critical steps for ensuring your (semi) quantification is accurate. 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. Even though western blot quantification is only semi-quantitative, you still need to be rigorous in how you quantify your blots to ensure your results are as accurate as possible. 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.
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