Protein Quantification Using ImageJ

Determining the concentration of protein in SDS-PAGE gel bands using ImageJ

• To determine protein concentration you will need to have a standard curve to compare your samples to

- For 5GB1, BSA works great as a protein standard, and a range of 0.025 μg/μL to 5.0 μg/μL works well as a range for the standard curve

• After running and destaining the gel, take a picture and save it as a .tif and as a .jpg (in case the tiff file can’t be opened—an issue I am experiencing at the other lab).

- Make sure you save your images as the same type of .tif each time!

1. Download the ImageJ software: http://rsbweb.nih.gov/ij/download.html

2. Open ImageJ

3. Go to File→Open→(your image)

• Does your image look too dark or too light?

- Image→Adjust→Brightness/contrast

• If image looks good...

4. Find the lane with the lowest concentration of BSA

5. Select the rectangle tool, and draw a box around the lane, making sure to include some of the empty gel between lanes and white space outside of the band

6. Go to Analyze→Gels→Select first lane

- A tiny “1” will appear in the lane

7. Make sure your cursor shows as an arrow, grab the rectangle you just made, and drag it to the next lane

- DO NOT DRAW A NEW RECTANGLE! You must drag the same rectangle you just made

- You want to compare the band in each lane using the exact same size/white space/noise as the originally defined area

8. Go to Analyze→Gels→Select next lane

- A tiny “2” will appear in the lane

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