Chemiluminescence is used to detect protein or DNA on blotted membranes. This detection method does not require any external excitation such as UV light.

The light emission is caused by a chemical reaction exciting a compound to a high-energy state. This unstable compound rapidly loses its excess energy by emitting a light. The emitted light is between 400 and 500 nm according to the substrate used.

The chemiluminescence chemical reaction occurs between an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) conjugated to an antibody.

The enzyme reacts with the chemiluminescent molecule (such as luminol or dioxetane) producing a weak signal. With the addition of an enhancer, the light intensity and the light duration increase.