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Image J Protocol

For Western blot analysis

2/10/2006

Terry D. Johnson

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1. Open Image J and, in it, open the .TIF file to be analyzed - in this case, “example.tif”.

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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.

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

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3. Select the Uncalibrated OD option from the Function: dropdown box, then click OK.

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4. A logarithmic plot should appear; move it to the side, but do not close the window.

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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.

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6. Carefully outline the first lane with the same tool. Go to Analyze > Gels > Select First Lane.

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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.

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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.

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(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.

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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.

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10. Once you have selected each band, you can analyze the area measurements in your spreadsheet of

choice.