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So, when I tried to draw a bigger rectangular this problem stopped! Of course the width of your rectangular should be made according to the band size, so change the height and make it a little taller. I had the same problem with Imagej and the solution came by coincidence! I realized that if I draw a rectangular with a width that is longer than twice the size of the height, then this thing happens. I have 1.41 version and there is no Outline Lanes in Gel Analyzer Options.ĭoes anabody know how to fight this problem? Maybe someone has an earlier version and can send it to me I have to say that sometimes it does work properlý, but mostly it does not and I do not see any order in this. Unfortunately ít stays not on the second band but jumps back to the first. Looking at your western image, if the bands are not linking with each other, there should be obvious trough at the empty area between the two bands, and you can draw boundaries to isolate the only. In order to achieve quantitative densitometric data from western blots, determination of the appropriate total protein load assures optimal dose-dependent responses per target (Figs. One broadly used program to quantify images of western blot bands is the Scion Image Software (Scion, Frederick, MD) Cite. 1.49K subscribers Subscribe Subscribed 152 Share 35K views 2 years ago Data Analysis Subscribe. Then I move the rectangular to the second band press 2 (or Ctrl 2, works the same). HOW TO QUANTIFY WESTERN BLOT BANDS using ImageJ Area Under The Peak Method Adwoa Biotech. Enlarge the image using the Magnifying Glass tool so you mainly just see the bands you want to measure. You have to do this again, because the powerpoint file saves it as an RGB tiff file. Convert the image to 8-bit (Image > Type > 8-bit). Figure 2 shows a stylised western blot of increasing concentrations of protein, and the signal intensity as measured by a. However, far more important is that you load the samples at least 3 times so you get an idea about loading and blotting variation (technical replicas). Open the TIFF file you created above in ImageJ. I open the file (.tiff), select rectangular, press 1 or Ctrl 1 to select the first lane. This represents a general problem of quantifying western blots with simple image analysis software, which may be unable to discriminate between similar-looking bands that have fallen off the end of the linear scale. This protocol offers an Image J macro/plugin that enable easy quantification of bands on western blots, dot blots, and fluorescent gels etc., by simply selecting bands with rectangle or. I have a problem with ImageJ analysing western blots.