Using ImageJ to Quantify Bands

Software

• ImageJ. Download or use applet. Both are available at http://rsbweb.nih.gov/ij/

Method

1. Save gel image and adjust to be vertically oriented
2. Using a rectangular box (box tool)select entire lane.
3. Select lane either by selecting Analyze->Gels->Select First Lane or press CTRL-1
4. Using side arrow key, slide box over to next lane and select Analyse->Gels>Select Next Lane or press CTRL-2
5. Plot lane by selecting Analyze->Gels->Plot Lanes or press CTRL-3
6. Draw baseline using the line tool. The baseline is to remove the "noise" from the background of the scanned gel.
7. If necessary use the line tool to connect peaks to the baseline. Use the line tool to connect the peaks to the baseline as they would if the peaks were individual and not connected in a line. Isolate individual peaks which protrude significantly from the baseline.
8. Select peaks using the wand (tracing) tool. Click on selected peaks (all peaks which are above the baseline) in order.
9. Go to the results window and the areas are calculated in numerical order. Be sure to count the peaks on the plot as to determine which peak corresponds to the peak of interest.
10. Copy the results into an excel file and analyse as necessary.