Image J Protocol

For Western blot analysis

2/10/2006
Terry D. Johnson
BE115

1. Open Image J and, in it, open the .TIF file to be

analyzed - in this case, example.tif.

BE115

1b. Optional
If the image is not already an 8-bit .TIF, go to
Image -> Type and select 8-bit.
If the image is not dark enough to analyze
properly, go to Image -> Adjust ->
Brightness/Contrast and adjust the slider
bars until the bands on the image are visible.
The pixel values will not be changed, just the
view.
BE115

2. In the main menu, select Analyze > Calibrate.

BE115

3. Select the Uncalibrated OD option from the

Function: dropdown box, then click OK.

BE115

4. A logarithmic plot should appear; move it to the side,

but do not close the window.

BE115

5. Go back to your .TIF image and select the Rectangle

Tool from your toolbox. Box all of the bands of interest
across all of the lanes and hit Crtl-H to bring up a
histogram.

BE115

6. Carefully outline the first lane with the same tool. Go

to Analyze > Gels > Select First Lane.

BE115

7. Drag the rectangle that you used for the first lane
over to the next, and mark it with Analyze > Gels >
Mark Next Lane. Dont draw a new rectangle! You want
every rectangle to have the same area. Repeat until all
the lanes have been marked.

BE115

8. Click on Analyze > Gels > Plot Lanes. Youll end up

with an image file with plots for each of the lanes that
you marked. The plot on top is the first lane that you
marked. Use Image > Rotate 90 Degrees Right (or
Left) and/or Image > Zoom > Out so you can see and
work with all of the plots.

BE115

10

(OPTIONAL) If you have more than one band per lane,

youll end up with a plot for a single lane that has
multiple peaks (one for each band).
Youll need to separate them into individual peaks with
the Line Tool as demonstrated below on another
image.

BE115

11

9. Select the Magic Wand tool and select each plot

beginning with the first. A box will appear with the Area
measurement for each plot as it is selected. The area of
that histogram plot represents the darkness of each
band in the gel.

BE115

12

10. Once you have selected each band, you can

analyze the area measurements in your spreadsheet of
choice.

BE115

13