Across blot comparisons - (Oct/12/2010 )

I have checked every resource I can find and have asked many post-docs, professors, graduate students, and undergrads from a variety of backgrounds including molecular biology, pharmacology, and neuroscience why across blot comparisons are not allowed with western blots and the only answer I get is, "Because there are so many variables that can affect the outcome of a western you can't compare two blots." Does anyone else but myself see a problem with this? It is illogical: I can't compare blots because I can't compare blots. Furthermore, there are an infinite number of variables that can affect any experiment. Using the western blot logic means that any experiment can never be compared with any other experiment, not even replications. So please, I ask all of you reading this post, please give me a logical and scientific reason why across blot comparisons cannot be made with western blots.

Halfro22 on Wed Oct 13 00:28:46 2010 said:

You can do across blot comparisons if the signal of interest shows an obvious-to-the-eye change in levels btwn experimental conditions. You really can't accomplish this accurately if the signal of interest is only marginally different (increased or decreased) but significant by way of densitometry. The biggest problem btwn across blot comparison is the exposure to film step...Using a phosphorimager can help to get around this.

One thing to do is to perform a densitometry analysis on one blot to determine the quantitative change in expression levels of your region of interest when compared lane-by-lane to a stably expressed control protein (e.g. GAPDH, tubulin, actin etc.). Do the same analysis on another blot to determine the quantitative change...if it's a robust and repeatable result, the blots might not look identical to one another, but the numbers from the densitometry analysis should be very close to one another.

you may also be able to compare blots if you have a reliable way to normalize the results.