Method for the validation by Western analysis of affinity reagents against post-translationally modified proteins. I. survey of existing antibodies, and II. development of method improvements

ABSTRACT The poor reproducibility of published research is a major focus of the scientific community in recent years. Western analysis Abs are often validated against antigen (Ag) materials such as mammalian cell lysates which have undergone treatment (e.g., chemical exposure, UV radiation etc.) to induce (or reduce) modification of residues on a protein of interest. Several stages during the generation of this material can pose challenges. First, it can be difficult to identify the appropriate treatment and cell line for the residue of interest. Second, treated cell lysates can be technically challenging to generate reproducibly. Even with careful adherence to a protocol, these lysates 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 validating PTM-specific Abs would be one where each Ag sample contains the target sequence residue with either 100% modification or 100% non-modification. We have developed a system that provides that certainty.