Quantifications of Western Blots with ImageJ

ImageJ is a powerful tool that can be used to compare the density or intensity of bands on an agar gel or western blot. In this tutorial, we will explore how to use ImageJ to analyze western blots and quantify the results.

To begin, open your digital image of the western blot in ImageJ using File > Open. The next step is to convert the image to grayscale by going to Image > Type > 8-bit. This is necessary because the gel analysis routine requires a grayscale image.

Once you have converted the image to grayscale, you can use the Rectangular Selections tool from the ImageJ toolbar to select individual lanes on your western blot. To do this, choose the tool and then click and hold in the middle of the rectangle on the first lane. Drag it over to the next lane and release the mouse button. The first lane will now be highlighted with a '1' in the middle.

After selecting the first lane, press the 1 key or go to Analyze > Gels > Select First Lane to set the rectangle in place. It is important to note that if you draw a short and wide rectangle, ImageJ will assume that your lanes run horizontally, which can lead to confusion. Instead, use a tall and narrow rectangle to enclose a single lane.

Assuming your lanes run vertically (with individual bands running horizontally), your rectangle should be tall and narrow. This is important because ImageJ assumes that your lanes run vertically by default.

Once you have selected all of the lanes on your western blot, you can proceed with quantifying the results using the Gel Analysis method outlined in the ImageJ documentation. This method provides a more accurate and reliable way to quantify the density or intensity of bands on an agar gel or western blot.

In conclusion, using ImageJ to analyze western blots is a straightforward process that requires some basic understanding of the software. By following these steps and using the Gel Analysis method outlined in the ImageJ documentation, you can accurately quantify the results from your western blots and gain valuable insights into the protein expression levels in your samples.

References

For further information on how to use ImageJ to analyze western blots, refer to the ImageJ documentation. Additionally, for a discussion of common mistakes that can be made during this step, see .

Quantifications of western blots with imagej