Virtual blots for teaching image analysis with ImageJ

SECTION 3

Measurements of bands with ImageJ: basic concepts

     In this section we will learn:

1) How to retrieve an image, and how to measure band intensities.

2) Analyse data using Excel

1) Measurement of band intensities.

a) Open file by clicking on the ImageJ toolbar File > Open  (and selecting the file I your computer), (Figure 2A).

Figure 2A

b) Convert the image to RGB stack by clicking on the ImageJ Toolbar Image > Type > RGBstack  (Figure 2B)

Figure 2B

c) Create a negative version of the file by clicking on the ImageJ Toolbar Edit > Invert ( Figure 2C)

Figure 2C

d) Sustract background by clicking on the ImageJ toolbar Process > Substract background (Figure 2D)

This sequence generates three versions of the original figure (shown in Figure 2A-D). For quantification of bands only the last version (Figure 2D) is used as follow:

Figure 2D

e) Select the “rectangular" selection tool from the ImageJ toolbar and draw a rectangle around the biggest band to be sure that the size of the rectangle will be big enough to measure all bands. In our example the biggest band is band 10 (Figure 3A).

Figure 3A

f) Drag the rectangle to the first lane and measure the intensity by clicking Analyze > measure (or press simultaneously Ctrl M). ImageJ will open a new “Result” window showing the values of each measurement (Fig. 3B)

Figure 3B

g) Drag the rectangle to the next band and repeat the measurement with all other bands (clicking Analyze > Measure (or press simultaneously Ctrl M).

Figure 3C

h) Repeat the measurement with all other bands (clicking Analyze > Measure (or press simultaneously Ctrl M).

Figure 3D

Export the results to Excel and analyze the relative intensity.

1) Make sure that the area measured (Column B) is the same.

2) Substract the intensity (RawIntDen, column H) corresponding to lane 1 (Control) for all bands (1-9). Write the final values in Column J (Blue).

In this case since we used a blot with 0% background the RaeIntDen value for line 1 is = 0.

Therefore, the values in column H are the same as the values for column J.

3) Write in Column K the values for the X axis (red).

4) Plot the data as follow: use column J for the Y axis and column K for the X axis.

The results are summarized in Fig. 4.

Figure 4

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