![]() Determine how much protein to load and add an equal volume 2X Laemmli sample buffer. H2AX antibody is used to identify DNA damage in the form of double strand breaks by. 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. Remove a small volume of lysate to perform a protein quantification assay. Purpose: This protocol describes how to quantify any type of cellular foci, such as phosphorylated Ser139 on histone variant H2A.X (H2AX) stained images of tissue or cells, using the free NIH Image Processing and Analysis in Java software called Fiji or ImageJ. 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. MUC2, p-PKC/ and p-Erk1/2 in TS-treated SL174T cells were quantified by using Image J program and statistical analysis was performed. 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. ![]() 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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