![]() ![]() 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. Their identity is confirmed by comparison to molecular weight markers (for size) and a positive control (size and signal). In the majority of cases, bands corresponding to the target protein will become visible upon treatment of the blot with substrate. 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 data produced with a Western blot is usually quite easy to interpret. Higher variability in WB than in IF among labs. Image Studio Software can be used for quick image acquisition for a variety of assays from LI-COR imaging systems, including Western blots, and for signal quantification and analysis of images from In-Cell Western and other assays. 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. Could we robustly and reproducibly quantify pre- to post-treatment. In fact, the gel for the wild type was accidentally loaded with more of the sample. Determine how much protein to load and add an equal volume 2X Laemmli sample buffer. 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. Remove a small volume of lysate to perform a protein quantification assay. ![]()
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