用ImageJ对WesternDNA和Blot图片灰度分析.docVIP

用ImageJ对Western Blot图片灰度分析 教程收集于互联网 来源中生网　　The good news is that even if you dont 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. Well start the ImageJ section by duplicating the method outlined above for Photoshop.　　1. Open your file.　　2. Under ImageType click on 8-bit to convert the image to grayscale.　　3. Go to the menu ProcessSubtract Background. Try a rolling ball radius of 50. This removes some of the background coloration from your image.　　4. Go to AnalyzeSet Measurements, and click the boxes for Area, Mean Gray Value, and Integrated Density.　　5. Go to AnalyzeSet Scale, and enter pixels in the box next to Unit of length.　　6. Go to EditInvert (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