Western Blot Data Analysis - (Mar/25/2010 )
I am wondering how people analyze their western blot data after they have their bands. Most specifically, I'm curious as to the exacty statistical analysis that are performed, whether log transformations are performed, normalizing samples to either housekeeping proteins or total proteins in the case of phosphoproteins. Any help would be appreciated!
You can analyse protein changes from westerns using densitometry - however it comes with a few caveats.
You can not compare between gels (i.e. you can't probe for house keepers on one gel and gene of interest on another), even if you poured, ran and transferred them at the same time - small variations in any conditions can have a big impact on the end result. This means you need to run you controls and samples on the same gel.
Yuu must not overexpose - saturation means that you have overexposed and will render the results un-useable.
If you wish to compare results normalised over several gels, you must have exactly the same exposure times for your gene of interest on every image, and exactly the same exposure times for each house keeper (e.g. actin) image.
The techniques is not sensitive - you can detect fold changes (e.g. 4 fold increase, means gene is upregulated 4 times over controls), but you can't say 1.2x, despite what your numbers are telling you.
I use Image J to analyse my WB bands.
In the program they have a gels command under the analyse section. You draw a rectangle outline across one lane and copy it across all your lanes. (This means you can measure the intensity of several different sized bands in the one lane).
Then you select plot lanes. This will give you a histogram of the different bands in each of your lanes. You then measure the area under the peak as the intensity of the band.
I concur with what bob1 has said regarding normalising to total protein (for phospho blots) or actin (for other blots).
I would do my stats looking at the fold change (say treated compared to untreated) across several gels on several different days.