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I have done a western blot and I want to remove the background signal when doing densitometry analysis of my protein bands of interest. I have read some articles online such as this one regarding high background in western blots. I have learnt that there are many factors that can contribute to high background in a western blot. However, I am still not sure what exactly is defined as background. Does it refer to non-specific antibody binding on the membrane, or can it also refer to regions on the membrane where there is no binding to any protein?

Any insights are appreciated.

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In this context (and many others), the terms "background signal" and "noise" can almost be used interchangeably, if that helps.

Generally speaking, noise or background refers to any signal that isn't coming from the thing you're trying to detect (typically in reference to how that signal might interfere or be confused with your "true" signal). So non-specific binding could qualify as background, but it can also refer to regions of the membrane that shouldn't have any protein binding (with any number of causes). One exception I can think of in practice is if you have a specific non-target band that consistently shows up, that might technically fit the definition (sensu stricto) of "noise", but a molecular biologist will more likely refer to it as a "technical artifact" rather than "background".

That said, there are still a number of different kinds of "noise" or "background" that may show up on western blots, and the article you referenced does a pretty good job of describing them, but it could benefit from including some images. Here's a similar guide with more images and links to other Western troubleshooting guides. Hope it helps.

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