# Dot Blot Analysis

> There are two methods for analyzing a dot blot in ImageJ. The first is to treat each row as a horizontal "lane" and use ImageJ's gel analysis function. The second is to subtract the background and measure the integrated density of each dot.

> Figure 1. Original dot blot.

> This dot blot image is available in the File/Open Samples menu in ImageJ 1.33s or later.

> Figure 2. Profile plots.

> This is what you get when you treat each row in the dot blot as a horizontal "lane" and use the gel analysis procedure in the ImageJ manual. The numbers on each peak are the size of the corresponding dot as a percentage of the total size of all the dots. "Invert Peaks" in the Analyze/Gels/Gel Analyzer Options dialog was enabled to avoid having upside down peaks.

> Figure 3. Profile plots of first row.

> In the second method, you measure the integrated density of each dot by outlining it and using the Analyze/Measure command. This method usually requires background correction of the image, which can be done using the Process/Subtract Background command. Figure 3 contains profile plots (Analyze/Plot Profile) of the first row of dots before and after background correction was done using the Subtract Background command with the rolling ball radius set to 25 pixels.

> Figure 4. Circular selection and integrated density measurements.

> After correcting the background, enable "Integrated Density" in Analyze/Set Measurements, create a circular selection, drag it over the first dot, press "m" (Analyze/Measure), then repeat for the other 27 dots. Notice how the image now has a black background? It was inverted (Edit/Invert) so background pixel values are near zero, which is required for correct calculation of integrated density. You can invert the lookup table (Image/Lookup Tables/Invert LUT) to restore the original appearance of the image. The "Use Inverting Lookup Table" option in Edit/Options/Image will invert the pixel data and invert the lookup table.

> Note that the "Integrated Density" option in Analyze/Set Measurements was added in ImageJ 1.33r.

> Figure 5. Results using plots (A) and integrated density (B).

> The first column in this spreadsheet contains the integrated densities (as a percentage of the total) of the 28 dots measured using the gel analysis procedure at rsb.info.nih.gov/ij/docs/menus/analyze.html#gels. The second contains the results obtained by doing background correction using Process/Subtract Background, selecting "Integrated Density" in Analyze/Set Measurements, and then measuring each of the 28 dots using a circular selection.

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