I want to check my protein expression but the sample (target protein) on my SDS PAGE shows a little difference as compared to the control. I measured the band density through IMAGE J but the band that appears darker shows less mean value than the control.
is there a better software of am I using it wrong?
Steps that I follow:
1. Upload the image and change it to greyscale.
2. adjust brightness and contrast if needed.
3. Invert the image if the bands are too dark , the bands will turn white and the background will be black
4. Make rectangular selections, analyze and measure each band one by one.
5. Higher the mean value, higher is the band density.
Dear Aqsa Ahmed
i tihnk that the problem could be related to the fact that you invert the colours in the step 3. Try to repeat the analisys avoiding this step.
in the following link you can find an example of my analysis approach.
https://proteocool.blogspot.com/2021/09/tips-of-week-13-densitometric-protein.html
best
Manuele
I like ImageJ. It puts a number on the bands' intensity, but it won't tell you anything that you couldn't guess already by looking at the gel. One thing that you can try is changing the destain time (if you still have the gel). You will see that the small bands lose intensity faster during destain than the large bands do - there may be a window of destaining time where your band of interest does appear more intense than the others in ImageJ. What are you using to express your target protein? Maybe it isn't effective.
There are some caveats for quantitation of bands in ImageJ (or any other software).
You need to work with 16-bit images, otherwise your range of quantitation will be too small (i.e. 256 greyscales in 8-bit images are pretty much useless).
Quantitation should be done with images where you measure the light (fluorescence or luminescence) not bands that are stained with dyes or silver. The staining on the gel surface and inside is most likely not homogeneous and there is a small linear range for this kind of images, even with 16-bit images (the stain would need to have some level of transparency to discriminate more or less dark areas, otherwise black will be black, no matter how much your band contains, the area of bigger bands will most likely not show the correct ratio of more or less protein)
Ideally, you should use a dilution series of the same (at least similar in size and staining) protein to check if your bands are in the linear range of the intensities.