Densitometry using ImageJ

Sample preparation

Convert the image to 8-bit using ImageJ function (Image→Type→8-bit).

Procedure

Use the square selection tool to highlight the first lane. Press Ctrl and 1 to set first lane (Command and 1 on the Mac).

Click the centre of the square and drag it across to the next lane. Press Ctrl and 2 to set the next lane. Continue this for the subsequent lanes (pressing Crtl and 2 every time).

For the last lane, repeat the procedure but press Ctrl and 3 to set the last lane. A window with lane plots will appear.

Use the line tool to draw the lines to eliminate the lane background from the calculations. Draw the line at where the peak begins and ends (bend in the line) for each peak.

Go to: Analyse→Gels→Label Peaks to get the report.

Alternatively, use the magic wand tool to highlight the area underneath the peak for each lane. The report will automatically pop up on the side.

Normalising the data

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. 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.