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hello and welcome to the James Lab tutorial on immunoblot densitometry I have a nice example of the two color like or fluorescence immunoblot here and Iamp;#39;m going to use to demonstrate how to do very careful quantitative estimation of the abundance of proteins and different samples what Iamp;#39;m going to start off with is the set of raw files that come off the like or Odyssey instrument first thing to emphasize is that all of the quantitative analysis should be on the raw 16-bit images that by default are saved as 700 and 800 TIFF files by the Odyssey software any of these other logs or JPEGs are colored or processed displays of the raw information so we want to make sure that weamp;#39;re always working with the 700 and the 800 Tiffamp;#39;s and then we keep track of what antibodies were monitoring in the 700 or 800 fluorescence channels Iamp;#39;m going to take these two images and open them in image a we do all of our quantification in image shade because it can appropri