![]() Divide IntDen for protein of interest by IntDen for beta-actin or house-keeping gene. 10.Save the table (will open in Excel) 11.Normalize the intensity: a. Hit M on the key board after selecting the area to see the measurements. 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. Select area you want to measure (keep it tight) 9. 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. 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. 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. A brief tutorial showing how to create a figure for publication or reporting using western blot (or other) images in Microsoft Word.All footage is the proper. 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. Use the square selection tool to highlight the first lane. Before doing Integrated density measurement in image J software is iit necessary to invert the Western blot image (from black protein band and white background to white protein band and black.
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