Fiji Gel AnalyzerChong Zhang

chong.zhang@bioquant.uni-heidelberg.de

Basic steps

1. requires the image to be a gray-scale image

2. Use Rectangular Selections tool to draw a rectangle (should be tall and narrow) to enclose a single lane. ImageJ assumes that your lanes run vertically (so individual bands are horizontal).

3. First rectangle: press the 1 key (Command + 1 on Mac) OR Analyze>Gels>Select First Lane

4. Next lane: mouse click + hold in rectangle & drag it OR move arrow keys (automatically line up on the same vertical axis), then Press 2 (Command + 2 on Mac) OR Analyze>Gels>Select Next Lane

5. Repeat Steps 3 + 4 for each lane

6. Profile plot: press 3 (Command + 3 on Mac) OR Analyze>Gels>Plot Lanes. Higher peaks represent darker bands. Wider peaks represent wider bands.

7. close off the peak: Straight Line selection tool

8. Measure: With the Wand tool, click inside the peak & repeat this for each peak, measurements (related to enclosed area, i.e. related to sum of intensities in the band) will shown in Results window

9. Analyze>Gels>Label Peaks: labels each peak with its size, expressed as a percentage of the total size of all of the highlighted peaks.

10. In Results window, select Edit>Copy All, content can be pasted to a spreadsheet.

Check points for image quality before analysing it:

� Do not saturate/clip image

� try to remove uneven background

Max pixel intensities

from these arrow-

indicated bands are

255 -> Image is

saturated!!! NOT

GOOD!

Please make sure

image intensities do

not reach to the max

and min values of

the image type

range! For example:

if you acquire 8bit

image, the intensity

range is [0-255]. So

the best is to acquire

images with min a

little higher than 0

and with max a little

lower than 255.

Little peak on the histogram indicates saturation!

Image courtesy: Kathleen Boerner

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Line profiles show that background has higher intensities in center & lower

towards border -> Image has uneven background!!! NOT GOOD!

We could subtract background using image processing methods.

Image courtesy: Kathleen Boerner

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Background subtracted image

Profile plot from Fiji

Gel output

Image courtesy: Kathleen Boerner

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Original image (F. A. Ran et al. Nature Protocol 2013)

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The elevated valley indicated in the plot is due to the

close distance between the two bands.

The easiest option is to try to set them more apart on

the gel

or one can try to approximate individual curves.

Background subtracted image

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Data analysis

1. Density of the peaks are relative (within the context of the set of peaks selected on the single gel image)! These values do not have units of µg of protein or any other real-world units.

2. To express them relative to some standard band that also present&selected. e.g. divide the Percent value for each sample by the Percent value for the chosen standard

3. To compare the density of samples on multiple gels or blots, the same standard sample should be present on every gel to provide a common reference

4. To test for significant differences between treatments in an experiment, quantify all using the same method in Relative Density. Also try to ensure that there is homogeneity of variance (of Relative Density) among the different treatments.

5. One can include a set of serial dilutions of a known standard on each blot.

Also, using the serial dilution curve, it is possible to express your sample bands in terms of the amount of protein.

Ref: http://lukemiller.org/index.php/2010/11/analyzing-gels-and-western-blots-with-image-j/

• four replicate samples of protein (four pipette loads out of the same vial of homogenate), so we expect the densities in each lane to be equivalent.

• upper row: protein of interest.

• lower row: loading-control protein*. In reality, the size and intensityvaries (i.e. 1 & 2 appear equivalent, 3 has half the intensity, 4 has half the intensity and half the size. Thus we need normalization/correction so as to compare the upper row bands.

One (synthetic) example (http://lukemiller.org/index.php/2010/11/analyzing-gels-and-western-blots-with-image-j/ )

*This loading-control protein is a protein that is presumably expressed at a constant level regardless of the treatment applied to the original organisms.

1 2 3 4

Snapshots of steps

4 lanes are roughly equivalent!

Blot-blot example(http://lukemiller.org/index.php/2010/11/analyzing-gels-and-western-blots-with-image-j/ )

Reference

• http://lukemiller.org/index.php/2010/11/analyzing-gels-and-western-blots-with-image-j/