![]() ![]() Below is an example of a Western Blot with six lanes showing the calculated Percent.Western blotting (WB) is an antibody-based experimental technique used to detect and quantify target proteins, which are often within a complex mixture extracted from cells or tissue. In otherwords, add up all the densities for a row of bands, then calculate what percentage of the total density is each band. For each band in a row, calculate the "percentage" of the total area for each band.You should now have an excel spreadsheet containing the "Area" of each band in all your images.You can save the Plot image, if you want, and copy it to the powerpoint that has the blot figure.After clicking in all the mountains, copy the data to an excel spreadsheet.As you click on the subsequent mountains, Image J will add another measurement to the window. The first time you click inside a mountain, ImageJ will open a new window and display the area inside the mountain. Click on the magic wand in the ImageJ tool bar, then click once inside each mountain. Calculate the area inside the mountains using the magic wand.If this is the case you need to re-make your images following the instructions above. If the bottom of the mountain does not extend to the bottom of the window, then you did not prepare your images correctly, and there was no white space above or below the band. The mountain represents the density of the band. Inside each window will be a mountain, unless there was more than one band in the lane, in which case there will be an equivalent number of mountains. ImageJ will open a new window that has windows stacked horizontally, with one window for each of your lanes. After you have dragged the last rectangle over the last lane, indicate that you are finished by selecting: Analyze > Gels > Plot lanes (or command -3). ![]() (Note: You don't have to be careful about the height of the boxes, as ImageJ will automatically align the boxes.) Repeat until all the lanes have been highlighted with a rectangle.Indicate this is another lane by selecting: Analyze > Gels > Select next lanes (or command -2) Using the mouse, click inside the rectange and drag (a second box will drag) to the second lane.Indicate that this is the first lane of the gel by selecting: Analyze > Gels > Select First lane (or command - 1) ![]() If there are multiple bands in the lane, make sure there is white space between the bands. Include some white space above and below the band (or bands).
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