用ImageJ对Western Blot图片灰度分析

用ImageJ对Western Blot图片灰度分析

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The good news is that even if you don't have access to a photo editing program such as Photoshop, you can now do all the same analyses using free programs. My favorite option is the freely available ImageJ from the National Institutes of

Health.

The homepage for ImageJ is here: /ij/index.html wherein you can find links to the download, documentation, additional plugins and so on.

Once ImageJ is installed, open it up and open your scanned film file. We'll start the ImageJ section by duplicating the method outlined above for Photoshop.

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. Go to Analyze>Set Measurements, and click the boxes for Area, Mean Gray Value,

and Integrated Density.

5. Go to Analyze>Set Scale, and enter "pixels" in the box next to Unit of length.

6. Go to Edit>Invert (or hit Ctrl+Shift+I) to invert the colors on the image. Now the dark areas are light, and the light areas are dark. As outlined above, this has the benefit of making the measured values for bands increase with increasing

protein expression.

7. Choose the Freehand Selection tool from the tool palette.

8. Draw a line around the boundary of your first band. As above, you need to use your own judgement about where the edges of the band are, and what is simply

background noise.

9. Hit the m key to take a measurement of the enclose area that you selected. The Results window should pop up, and each of the measurements you selected in step 4 should appear. Note that the Integrated Density column is simply the Area and

Mean Gray Value columns multiplied together.

10. Use the Freehand Selection tool to select the next band, and press m to take the measurement. Repeat this for each of you bands, including the standard.

11. When you are finished, you can go to the Edit menu in the Results window,

and choose Copy All. You can then paste the results into a spreadsheet for later

use.