MILKSHAKE Western blot and Sundae ELISA: We all scream for better antibody validation.

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.