Quantifying Western blots - (May/08/2009 )
I Use ECL PLus detection for my Western blots and do the imaging with a CCD camera (Fuji LAS-1000 or Fuji LAS-4000). I then quantify the images using ImageQuant software from GE.
Does anyone know if there is a big difference between the various softwares used to quantify Western blots? I'm particularly interested in comparisons between ImageQuant, Image J and Scion Image, since those are the softwares used at the institute where I work.
Is it OK to use different software to quantify data that will end up in the same paper? Do you usually list the software used for quantification in your publications? And finally, when you retrieve images with your camera, is it important to save the files as raw data in form of .img files (in the case of Fuji cameras) or can they be saved directly as .tiff files?
If your quantitation is performed correctly there should be no difference between the measurements gained from different software, so long as the units are consistent. If there are, you need to look at how each software package handles the analysis and make sure that each is doing it the same way. Ideally you would only use one method for consistency.
.TIFF files should be fine, however the maximum amount of data is always found in the camera specific file type such as .RAW. You will have to convert camera specific file types to usable files, which should always be .TIFF for publication.