Quantification of protein bands using densitometry

Overview

Follow this protocol to quantify protein bands using densitometry. The protocol is based on the excellent instructions for using ImageJ to quantify bands written by Luke Miller, November 2010 http://www.lukemiller.org/ImageJ_gel_analysis.pdf. To perform this analysis you will need the ImageJ software, which can be downloaded from the NIH website

Protocol

Preparing your band image file:

• Open the image using imageJ
• Save the files as TIFF file using a different name than the original
• Convert the image to 8-bit (Image > Type > 8-bit)
• If you have black background and white bands, invert the image (Edit > Invert). Otherwise leave as is.
• Rotate the image so the bands are lined up horizontally (Image > Tranform > Rotate)
  • Select the angle you want (it will rotate clockwise)
  • Select at least 6 gridlines to help you judge whether or not the bands are horizontal
  • Using "Bilinear" interpolation
  • Check the "Fill with Background Color"
  • Check the "Preview"
  • Hit OK
• Save the file again
• If there are more than one set of bands that you want to analyze, make as many copies of the file as there are bands that you want to analyze.
• For each band file do the following:
• Enlarge the image using the Magnifying Glass tool so you mainly just see the bands you want to measure
• Crop the image by highlighting the bands you want to measure using the Rectangle tool. Try to draw the rectangle as small as possible around the horizontal line of bands. Then crop (Image > Crop). You should now have a very small file with a rectangle that only includes the bands you want to measure
• Save the file, and repeat with the other file copies, if you have more than one set of bands on the same original image.
• Repeat this process using your loading control image file, if it is a different file.
• Open a Powerpoint file and drag all your image files into powerpoint
• Line them up, leaving white space in between each one.
  • Note: You can also label everything, put in ladder images and other things, as long as you leave white space around the images.
  • Do not change sizes or adjust the images in anyway. The lanes should line up.
• Save the file as a TIFF file using Powerpoint save as command. Powerpoint saves a folder with the filename, and then saves each slide as slide1.tiff, etc.

Measuring band densities.

• Open the TIFF file you created above in ImageJ.
• Convert the image to 8-bit (Image > Type > 8-bit). You have to do this again, because the powerpoint file saves it as an RGB tiff file.
• Enlarge the image using the Magnifying Glass tool so you mainly just see the bands you want to measure
• Use the rectangle tool to create a vertical rectangle in the first lane. Include some white space above and below the band (or bands). If there are multiple bands in the lane, make sure there is white space between the bands. Indicate that this is the first lane of the gel by selecting: Analyze > Gels > Select First lane (or command - 1)
• Using the mouse, click inside the rectange and drag (a second box will drag) to the second lane. Indicate this is another lane by selecting: Analyze > Gels > Select next lanes (or command -2)
• Repeat until all the lanes have been highlighted with a rectangle. (Note: You don't have to be careful about the height of the boxes, as ImageJ will automatically align the boxes.)
• 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). ImageJ will open a new window that has windows stacked horizontally, with one window for each of your lanes. 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. The mountain represents the density of the band. 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. If this is the case you need to re-make your images following the instructions above.
• Calculate the area inside the mountains using the magic wand. Click on the magic wand in the ImageJ tool bar, then click once inside each mountain. The first time you click inside a mountain, ImageJ will open a new window and display the area inside the mountain. As you click on the subsequent mountains, Image J will add another measurement to the window.
• After clicking in all the mountains, copy the data to an excel spreadsheet.
• You can save the Plot image, if you want, and copy it to the powerpoint that has the blot figure.
• Repeat the process with each image.
• You should now have an excel spreadsheet containing the "Area" of each band in all your images.

Calculating relative densities

• For each band in a row, calculate the "percentage" of the total area for each band. In otherwords, add up all the densities for a row of bands, then calculate what percentage of the total density is each band. Below is an example of a Western Blot with six lanes showing the calculated Percent.

• Next, calculate the relative density of each band, using your control condition. Divid the percent of the condition band by the percent of the control band. The table below shows the relative density based on the percent column. Note, by definition, the Control condition relative density should always be 100.

• Next, repeat the process for your loading control bands. Below is an example that goes with the above table.This is NOT a good representative Western Blot because, as you can see by the numbers below, the loading control bands varied quite a bit in density. But that's why we use a loading control.

• For the last step, calculate the adjusted relative density for your bands of interest. To do this, divide the relative density of condition 1 using the experimental antibody, with the relative density of condition 1 using the loading control antibody. In the case above, this would be 46 divided by 54.

• You are done! You can use a bar graph to visually display the adjusted relative density. Note the control condition, again by definition, should always be 100. In the example above, the protein being measured was severely decreased in condition 4, but relatively unchanged in the other conditions. Also remember that there are no units attached to these numbers, they are all relative and they cannot be used to indicate an actual protein amount.