Pan-Specific Antibodies as Novel Tools to Detect Valyllysine

Abstract

Objective: Amino acyl modification of lysine residues is an essential mechanism of nutrient sensing that regulates various biological functions including reproduction. At present, the lack of pan-specific antibodies for a recently identified lysine valylation hinders the characterization and detection of this modification. The objective of this study is to raise pan-specific antibodies that may facilitate the identification of novel expression patterns of lysine valylation. Methods: Chicken ovalbumin was valylated as an immunogen to raise polyclonal antibodies (PcAbs) in rabbits. The population of the pan-specific antibodies recognizing valylated lysine was purified using the chemically synthesized valylated peptides consisting of random amino acids. The specificity of the antibodies was evaluated using ELISA, dot blots, Western blots, and immunohistochemistry (IHC) staining in human epididymis as well. Results: A preliminary and simple strategy to make an anti-valylated lysine PcAb was developed. The recognition of the antibodies to valyllysine was evaluated as pan specific. This was useful for the detection of the newly identified valyl modification using ELISA, dot blots, and Western blots. The antibodies were also successfully utilized in IHC assays, which revealed novel valyllysine modification patterns in epididymis tissues of human. Conclusions: A new antibody tool was provided for the study of lysine valylation. The novel expression patterns of valyllysine in the epididymis suggest that this modification may be involved in sperm maturation.