![]() Since the area intensity is in arbitrary unit, it can also be normalised to the BCA assay measurement, DNA content or any other number chosen. To normalise the intensity of the area underneath the peak to the Ponceau staining, measure the intensity of 3 randomly chosen peaks on the Ponceau image, average the measurements and use that value to normalise the data against. The report will automatically pop up on the side. HOW TO QUANTIFY WESTERN BLOT BANDS using ImageJ Area Under The Peak Method Adwoa Biotech. Purpose: This protocol describes how to quantify any type of cellular foci, such as phosphorylated Ser139 on histone variant H2A. The same technique can be used for quantification of DNA or RNA from films. The quantification will reflect the relative amounts as a ratio of each protein band relative to the lane’s loading control. Go to: Analyse→Gels→Label Peaks to get the report.Īlternatively, use the magic wand tool to highlight the area underneath the peak for each lane. This protocol will allow you to relatively (no absolute values) quantify protein bands from western blot films. ![]() Draw the line at where the peak begins and ends (bend in the line) for each peak. Use the line tool to draw the lines to eliminate the lane background from the calculations. Continue this for the subsequent lanes (pressing Crtl and 2 every time).įor the last lane, repeat the procedure but press Ctrl and 3 to set the last lane. Press Ctrl and 1 to set first lane (Command and 1 on the Mac).Ĭlick the centre of the square and drag it across to the next lane. The measurement of the areas will be bumped to a 'Results' window. Continue selecting the area outlines of the remaining lanes. Use the square selection tool to highlight the first lane. On the ImageJ interface, select the 'magic wand' button and then click on the line defining the area of the curve of the first standard, and the areas of the curves in your protein analysis lanes.
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