Fiji Gel Analyzer Chong Zhang [email protected] Basic steps 1. 2. requires the image to be a gray-scale image 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 One example Image courtesy: Kathleen Boerner Max pixel intensities from these arrowindicated bands are 255 -> Image is saturated!!! NOT GOOD! Little peak on the histogram indicates saturation! 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. One example Image courtesy: Kathleen Boerner 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. One example Image courtesy: Kathleen Boerner Background subtracted image Profile plot from Fiji Gel output Another example Original image (F. A. Ran et al. Nature Protocol 2013) Another example Background subtracted image 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. Data analysis 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. 1. Ref: http://lukemiller.org/index.php/2010/11/analyzing-gels-and-western-blots-with-image-j/ One (synthetic) example 1 2 3 (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 intensity varies (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. *This loading-control protein is a protein that is presumably expressed at a constant level regardless of the treatment applied to the original organisms. 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-westernblots-with-image-j/