Western blot quantification with imagej4/15/2024 Like Amanda mentioned all of the quantification goes back to the significance and the magical p<0.05! However, seeing is believing so when we see that your lane 1 has a thinner band when compared to lane 6 can we take that as experimentally relevant? Or will we always be trapped in the paradigm of significance. Even though differences in the amount of protein can sometimes be detected with the naked eye quantification with the software we must be aware of the bias we introduce. Even though the lanes are parallel once you take the picture you have to take into account the noise, the amount of protein loaded into your gel and as you mentioned the bias created when you are responsible for selecting the correct area for the quantification. We recently had to perform a couple (always trying to please grant reviewers) and while I was analyzing the data using ImajeJ I started to wonder the exact same thing. I find this post extremely interesting, my lab is a physiology lab and we try not to do western blots unless we absolutely have to. Since your analysis says there is, it is important to know if this difference is biologically relevant. Again, this is all just me observing this band without knowing the scientific relevance (or really the experiment at all), but when I look at those bands I do not see a difference in intensities. This also brings to light a point that sometimes we rely to heavily on the "statistical significance" of these band intensities. Or if it would just be another layer of area selection bias. Me wonder if the loading control would help. Regardless your analysis could shows the consequences of these types of analyses, and it makes I realize this was just a quick experiment of intrigue, and it looks like maybe that top band is the control, or a spurious band. I am curious if you did the same analysis with a loading control, and then normalized the signal. Something like a technical replicate of the area selection? As you mentioned, it is important to correct for noise and that is another added step of variance and potential bias. It makes sense that there is variation between your attempts because of the area around the band, and I wonder if it would be possible to take that into consideration when you are doing western blot analysis. This is a really interesting analysis, that I have always wondered about.
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