![]() You'll just have some variation in your normalized sample-control values. This is similar if all the biological replicates are on one membrane. Normalize the sample-control normalized values to your loading control or total protein controls, respectively.Repeat this for your loading control bands, or total protein bands if looking at post-translational modifications. This value will become "1" and every other band will be some fraction or multiple of it. For example "Untreated" or "wild-type" lanes can serve as a normalization standard. Normalize each area to the area of a sample-control band.Export your AUCs to MATLAB or Excel, or whatever program you like to crunch numbers in.Use the wand tool to measure areas under the curve (AUCs).This step encloses a baseline-normalized peak in the same position for each lane (if the peaks don't line up well you may need to go back to step 5 to rotate your bands, or you might need to approximate the base of each peak individually. Now draw a rectangle vertically across the plotted lanes window such that the left and right borders of the rectangle encompass the left and right boundaries of each peak, and the top/bottom of the rectangle enclose all peaks. Your peaks should be baseline separated, or very nearly so due to background subtraction.Use Tools>Gels to select the first lane (Ctrl 1), and then Ctrl 2 for each subsequent lane.Again use Selection>Specify to draw a vertical rectangle slightly wider than your band size, and with a length long enough to capture the whole lane (in case any smaller/larger bands are present).This is important for making your area under the curve measurements below, as it will keep your peaks lined up nicely at the "plot lanes" step. ![]()
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