It is worth reiterating that the “area” values and percentages reported by Image-J are always relative to the total size and density of bands that you have selected in a particular image. ImageJ reports the same “area” value for both of these lanes. Compare lanes 1+3, which both have an equal number of gray pixels and equal gray values (i.e. Image-J also accounts for gaps in a band, as shown in Figure 27 above. In Figure 26 above, altering the shape of the band, but maintaining the same gray value and area (compare lanes 1+3) yields an equivalent value from ImageJ. The same holds true for bands of different shapes. By the same token, if you halve the gray value but maintain the same area (compare lanes 1+5), the value reported by Image-J is halved. When you halve the area of a band, but maintain the same gray value (compare lanes 1+2), the value reported by ImageJ is half as large. Note that these “area” values are a RELATIVE measure of the size and density of each peak you clicked with the wand tool. Additionally, I have included the “area” value returned by Image-J after plotting the bands and clicking in each peak with the Wand tool. The gray value and area of each band are listed above the band (in this case a lower pixel value = darker band). In Figure 25 above, I have drawn out a set of fake “bands” in Adobe Illustrator. The images below may help illustrate what Image-J is measuring. With regard to the ImageJ gel analysis routine, there has been some question of what the values reported by ImageJ correspond to. Because your relative density values are all normalized to the standard sample, you may wish to eventually re-scale your relative density values once again using your experimental controls’ average value as a baseline. Now you can finally begin calculating average relative density values for your experimental control samples (maybe these are samples 3+6) and your experimental treatment samples (perhaps these are samples 1+4 and 2+5) to carry out the real comparison of protein expression under your treatment conditions. At this point you can ignore the values of the standard samples, since you only needed those to ensure that your cross-gel comparisons were scaled properly. ![]() When finished, you’ll note that because every sample lane is normalized to the standard sample in Lane 1 on that particular gel, and every gel contains the same standard sample, your cross-gel comparison is already accomplished, since every sample is now normalized to the same standard sample (Figure 22).Īfter all of this work, you’re left with (adjusted) Relative Densities for each of your samples (the yellow cells in Figure 22). After you’ve scaled the density of the protein of interest for each sample to the standard sample, and adjusted the relative density based on the loading-control band, you are left with an estimate of the relative density of the protein of interest in each lane on your gel (relative to the standard in lane 1).
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