How to use Image J software in quantitative analysis of western blots

1. Open your file.

2. Under Image>Type click on 8-bit to convert the image to grayscale.

3. Go to the menu Process>Subtract Background. Try a rolling ball radius of 50. This removes some of the background coloration from your image.

4. Choose the Rectangular selection tool. Draw a rectangle around your first lane. Encompass some area of the lane above and below the band of interest. Press the 1 buttom (or go to Analyze>Gels>Select first lane). A new window will pop up with a copy of your image and a label over your first rectangular selection.

5. Use the arrow keys to move the rectangle over the next lane. Press 2 (or go to analyze>Gels>select next lane) to place a selection around the lane. Repeat this for each lane on the membrane, moving the box and pressing 2 to place the selection.

6. When finished, press 3 (or go to analyze>Gels>Plot lanes), which pops up a new window with a profile plot of each lane.

7. Choose the straight line selection tool. At the base of each peak, draw a line from one side of the peak to the other. This encloses the area of the peak. The tails to either side of the peak are the background signal.

8. When each peak has been closed off at the base with the straight line tool, choose the magic wand from the tool palette. Using the spacebar and mouse, drag the profile plot back down until you are at the top peak. With the wand, click inside the peak. Repeat this for each peak as you go down the profile plot. When each peak has been selected, go to Analyze>Gels>Label peaks. This labels each peak with its size expressed as a percentage of the total size of all the measured peaks. You can go to the results window and choose Edit> Copy All to copy the results for placing in a spreadsheet.