Western Blot scans need to be converted into grey scale and saved as a JPEG file.
1) Open Western scan in Image.
2) Click the 'rectangular selection' (under file menu) and select a box around a band of interest. Avoid any background if at all possible. The same box size must be able to fit all of the bands you would like to measure.
3) Once you have boxed an area go to the 'analyze' drop menu. Select 'measure'. A results box should pop up.
4) Use the arrow curser to move your box along to the next band of interest, and repeat the 'measure'.
5) Once you have finished measuring all protein bands, move the box onto the background. Measure the area of the background blot (in the same manner). If the background is somewhat blotchy and uneven in colour then select a background area equivalent to that around the protein band.
6) Once all the measurements are finished, select 'all' (control A) and 'copy' (control C) and paste in excel.
7) In excel erase all the values except the Mean values. Subtract all the Mean values from 255 (including the background). Then subtract the background from each band value. This will give you the band intensity;
Background mean = 189.527
Sample A, mean = 120.977
Sample B , mean = 109.877
Subtract from 255
Background mean = 65.473
Sample A, mean = 134.023
Sample B , mean = 145.123
Sample A = 68.55 = intensity
Sample B = 79.65 = intensity
8) Next we calculate the area of each protein band. Reopen in the blot in Image. Go the 'image' drop menu. Scroll down to 'adjust' and select 'threshold'. A threshold box will pop up (the blot may become red pixilated.). Drag the bar cursers to the far left (the blot should then be un-pixilated). Drag the cursor of the bottom bar slowly to the right, until the protein bands are all accordingly pixilated in red. Once the bands are fully filled in red, press the 'Apply' box then press the 'OK' box. Your bands should now appear as black on a white background.
9) Measure the protein area by selecting a rectangular box around a black band (you may adjust the box size between each band). Go to the 'analyze' menu and choose 'histogram'. A histogram box should pop-up. This will display values such as count, mean, std dev, min, max, mode. Ignore these!! Place your computer mouse curser over the Y- axis at 0. A 'value' and 'count' number will appear on the bottom of the box (only when the curser is held over the y-axis). Record the 'count' number in excel (this is the area of your band).
10) Record the area of each protein band in excel. Multiply the Intensity x Area which will give you the "integrated pixel value". ie.
Sample A = 68.55 (intensity) x 411 (count) = 28174.05
Sample B = 79.65 x 770 = 61330.5
The integrated pixel value is large, so we express it as 103.
Integrated pixel value x 103 Sample A = 28.1
Integrated pixel value x 103 Sample B = 61.3