Mendez, Q., Driscoll, H. A., Mirando, G. R., Acca, F., Chapados, C. D., Jones, K. S., Weiner, M., Li, X., & Ferguson, M. R. (2023). MILKSHAKE Western blot and Sundae ELISA: We all scream for better antibody validation. Journal of Immunological Methods, 521, 113540. https://doi.org/10.1016/j.jim.2023.113540
Knowing that an antibody's sensitivity and specificity is accurate is crucial for reliable data collection. This certainty is especially difficult to achieve for antibodies (Abs) which bind post-translationally modified proteins. Here we describe two validation methods using surrogate proteins in western blot and ELISA. The first method, which we termed “MILKSHAKE” is a modified maltose binding protein, hence the name, that is enzymatically conjugated to a peptide from the chosen target which is either modified or non-modified at the residue of interest. The surety of the residue's modification status can be used to confirm Ab specificity to the target's post-translational modification (PTM). The second method uses a set of surrogate proteins, which we termed “Sundae”. Sundae consists of a set of modified maltose binding proteins with a genetically encoded target sequence, each of which contains a single amino acid substitution at one position of interest. With Sundae, Abs can be evaluated for binding specificities to all twenty amino acids at a single position. Combining MILKSHAKE and Sundae methods, Ab specificity can be determined at a single-residue resolution. These data improve evaluation of commercially available Abs and identify off-target effects for Research-Use-Only and therapeutic Abs.