Year: 2026
Type: preprint
Abstract: This protocol describes the covalent conjugation of a mouse IgG antibody to horseradish peroxidase (HRP) using glutaraldehyde activation. The IgG was prepared by buffer exchange with PBS 1X using Vivaspin‱ 2 centrifugal filters (3 kDa MWCO), while activated HRP was similarly processed to remove residual free glutaraldehyde. Approximately 280 µL of IgG and 280 µL of activated HRP were subsequently combined at a 1:1 volume ratio for the conjugation reaction. Residual aldehyde groups were blocked with glycine, followed by purification and concentration using an Amicon Ultra 100 kDa centrifugal filter. The resulting IgG–HRP conjugate was formulated in PBS containing BSA, glycerol, and gentamicin and stored protected from light at 4°C. The original desalting step using a HiTrap Desalting Sephadex G-25 column was replaced with Vivaspin‱ 2, 3 kDa MWCO centrifugal filtration for IgG buffer exchange and removal of free glutaraldehyde.
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7204186236