The results of Western Blots can be assessed visually by making comparisons between bands in different lanes. However on occasion, these differences may be subtle and so a more quantitative method should be used.

ImageJ is a Java-based image analysis package widely used by scientists in quantitating visual results such as bands on gels or photomicrographs. It is available free for download from the National Institutes for Health (NIH) in the US. This Appendix will take you through the basics of how to use ImageJ in image analysis.

Selecting the area to be analysed

Open ImageJ from the Desktop or Program Menu. From the File menu, open the image file for the PCNA blot you obtained from the Chemidoc system.

Select a rectangular area around the first band using the “Rectangular Select” tool. The area selected should be about twice as tall as it is wide and should be as wide as the widest band on the blot.

ImageJ

Press “CTRL” “1” to mark this as the first band. The selection box should be yellow with a blue box with the number “1” inside, indicating that it is the first band selected.

Using ImageJ Figure 2

Move the mouse cursor so that it sits over one of the box border and changes into an arrow. Drag the box so that it sits over the next band and press “CTRL” “2” to select the next band.

Using ImageJ Figure 3

Repeat this step for each of the bands, pressing “CTRL” “2” for each subsequent band. With each selection, a small blue box should appear in each of the selection boxes indicating the number of the band (ie. 1-7 in our example).

Using ImageJ Figure 4

Once all of the bands have been selected, press “CTRL” “3”. This brings up another image which shows histograms indicating the intensity of each of the bands (the larger the histogram, the brighter the band). There should be a histogram for each of your bands – you may need to select the “drag” button and drag the image down to see all of them.

Using ImageJ Figure 5

Starting with the histogram for the first band, select the “draw line” button and draw a line across the top of the histogram from where it first begins to drop steeply until where it levels out again.

Using ImageJ Figure 6

Select the “magic wand” button and click anywhere inside the histogram. The selected area should be outlined in yellow and a new window named “Results” should appear which indicates the intensity of the band as a numerical value – the brighter the band, the higher the number.

Using ImageJ Figure 7

Repeat this process for each of the histograms. You should end up with a numerical value for each of the histograms representing the bands. Once complete, select “File” in the Results window and save the values as a spreadsheet. These values can then be copied into the “Blots Quant” spreadsheet for analysis and comparison.

Using ImageJ Figure 8


 

You will need to do this analysis for any other blots you have images for.