Protocol Online logo
Top : New Forum Archives (2009-): : Protein and Proteomics

Western blot data analysis - (Oct/11/2010 )

Hi everyone,

I have been trying to figure out the best way to analyze data from my western blots and have not found a good method or a standard method. Here is what I have been doing: I image my western with Kodak molecular imagining software and save the picture as a *.tiff. I then load that tiff image into imageJ (NIH). I then use the method described here ( to obtain the integrated density of each band. I input that data into Excel, average the integrated density of the experimental group and divided that value by the integrated density of the control groups. This will give me a percent change relative to the control group (I believe). This methods seems to work if I am comparing one experimental group to one control group. What if I have 3 experimental groups and 1 control group, would this method still work? If I run two gels, would I be able to statistically compare percent change relative to the control group? Or is it impossible to have across blot comparisons? Finally, what stats would I run? I'm assuming t-tests for 2 groups and ANOVAs for multiple groups. Any help would be greatly appreciated because I cannot find a resource anywhere that describes how to analyze western blot data. Thanks!


Disclaimer: I am not a fan of quantitative western, I don't think it works very well...

In my experience, even with the same lysates run on separate gels (made at the same time, run at the same time in the same buffer), blotted at the same time with the same buffer, there is a lot of inter-gel/membrane variation that is not really reproducible. I guess if you did many gels of the same things and averaged the results it would be OK as a method.

Within gels, the results are usually OKish, though small changes in the size of the band seem to influence the value you get out at the end of densitometry analysis. You need to be extremely careful not to over-expose bands, as this essentially takes you outside the "reference range" of densitometry.