be1151 image j protocol for western blot analysis 2/10/2006 terry d. johnson
TRANSCRIPT
BE115 2
1. Open Image J and, in it, open the .TIF file to be analyzed - in this case, “example.tif”.
BE115 3
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 7
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 8
6. Carefully outline the first lane with the same tool. Go to Analyze > Gels > Select First Lane.
BE115 9
7. Drag the rectangle that you used for the first lane over to the next, and mark it with Analyze > Gels >
Mark Next Lane. Don’t draw a new rectangle! You want every rectangle to have the same area. Repeat until all
the lanes have been marked.
BE115 10
8. Click on Analyze > Gels > Plot Lanes. You’ll 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 11
(OPTIONAL) If you have more than one band per lane, you’ll end up with a plot for a single lane that has
multiple peaks (one for each band).
You’ll need to separate them into individual peaks with the Line Tool as demonstrated below on another
image.
BE115 12
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.