I am trying to do Western blot quantification. I have read all the NIH documentation on this and several other sites. I am doing it using the rectangle tool followed by the wand quantification. Infuriatingly, I find that the size of the box that I draw affects the quantification, with or without background subtraction. There are also some weird 'scaling' effects. For example, if I draw boxes around my bands and then draw a box on the background in the same set, the background quantification is basically zero. If I draw a box only around the background, I see some jaggedy lines and if I quantify the area under these, it ends up being comparable to the area I get for the gel bands. If anyone knows what I'm doing wrong, I'd greatly appreciate hearing from you. This has been driving me crazy all day.