用ImageJ对Western DNA和Blot图片灰度分析

用ImageJ对W estern 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 don't have access t o 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 Institut es of Health.

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

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

1. Open your file.

2. Under Image>Type click on 8-bit t o convert the image to grayscale.

3. Go to the menu Process>Subt ract 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 Int egrated D ensit y.

5. Go to Analyze>Set Scale, and ent er "pixels" in the box next t o Unit of length.

6. Go to Edit>Invert (or hit Ct rl+Shift+I) t o 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 prot ein expression.

7. Choose the F reehand Selection tool from the t ool palette.

8. Draw a line around the boundary of your first band. As above, you need to use your own judgement about

where t he 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 select ed. The Result s window should pop up, and each of the measurements you select ed in step 4 should appear. Not e 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 st andard.

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 lat er use.

教你使用ImageJ分析电泳条带灰度比-ImageJ使用教程

ImageJ这套软件可以自动帮你你计算细胞数，也可以定量分析DNA电泳或是Western blot条带。

step 1.首先打开软件后，开启图档

step 2.请先做校正，选择Analyze底下的Calibrate选项，再选择校正的模式，使用Uncalibrate OD，再按ok

按下ok之后会出现校正的图形

Step 3.在要分析的第一条(first lane)加上一个长型框(工具列第一个选项)，再按下Analyze/Gels/select first L ane快速键(Ctr+1)，此时框架中会出现一个号码1，之后可以移动框架到第二个lane再选择Analyze/Gels/select second Lane快速键(Ctr+2)，当然可以一直加下去，最后按Analyze/Gels/plot Lanes快速键(Ctr +3)。

Step 4.分析以后会出现图型表示你刚选择的框内的影像强度，此时可以看到有几个比较高的区段，就是我们想定量的band，使用直线工具(工具列第五个选项)先将图形中高点为有band的区域和没有band的区域分开再，使用魔术棒工具(工具列第八个选项)点选要分析的区域。