Post translational modification Abs validated by surrogate protein Western blot.

PTM antibodies may not be mono-specific and may bind multiple modification types.

Surrogate protein ELISA reveals residue side chains’ impact on antibody binding.

Off-target antibody binding may be reduced using surrogate protein ELISA.

Thoroughly validated antibodies (Abs) are crucial for the generation of meaningful scientific data. Abs for post translationally modified (PTM) protein targets in particular pose added validation challenges. The MILKSHAKE method employs surrogate proteins which are either modified or non-modified at a specific site. Western blot is used to observe the binding of PTM Abs to the surrogate proteins, indicating the specificity of the PTM Ab under test. In this study, we expand the utility of MILKSHAKE by validating acetyl and methyl specific Abs and by introducing another surrogate protein antigen based on cellulose binding domain (CBD) to evaluate Abs in a single western blot lane. This study also explores the use of ‘Sundae’ surrogate protein ELISA specifically for therapeutic Ab evaluation.

Post translational modification

Antibody

Sortase ligation

Validation

Western blot

ELISA

© 2025 The Authors. Published by Elsevier B.V.