The His tag typically consists of 6 or 8 histidine residues. It can be fused to the N-terminus or C-terminus of a fusion protein. Due to its small molecular weight and ease of separation and purification, it is one of the most widely used systems in tag-based protein purification. Antibodies against the His tag enable precise detection and purification of His-tagged fusion proteins. Horseradish Peroxidase (HRP) is conjugated to the anti-His tag antibody through a specific process. In Western blot (WB) experiments, upon adding chemiluminescent substratesA and B (typically luminol and hydrogen peroxide), the luminol reacts with hydrogen peroxide under the catalytic action of HRP to form an unstable peroxide intermediate. This intermediate decomposes, generating an electronically excited species. When this species returns to the ground state, light is emitted, allowing for rapid detection of the His-tagged fusion protein.