The poor reproducibility of published research is a major focus of the scientific community inrecent years. Western analysis Abs are often validated against antigen (Ag) materials such asmammalian cell lysates which have undergone treatment (e.g., chemical exposure, UV radiationetc.) to induce (or reduce) modification of residues on a protein of interest. Several stages duringthe generation of this material can pose challenges. First, it can be difficult to identify theappropriate treatment and cell line for the residue of interest. Second, treated cell lysates can betechnically challenging to generate reproducibly. Even with careful adherence to a protocol, theselysates may have varying degrees of modification present in "treated" and "untreated" samples.This can lead to incorrect specificity determination for the Ab. A reliable method for validatingPTM-specific Abs would be one where each Ag sample contains the target sequence residue witheither 100% modification or 100% non-modification. We have developed a system that providesthat certainty. Public Health Relevance Statement:
Antibodies (Abs) are the most frequently used tools in science research and in clinical assays.
But there is still an absence of universally accepted guidelines or standardized methods for
validating these reagents. We have invented a surrogate validation method to validate antibodies
against post-translationally modified protein sites. To determine quantitatively just how bad the
problem may or may not be, we propose to test and validate the 10 most cited antibodies from
several different manufacturers. Project Terms: