Peer Review Report For: A guide to selecting high-performing antibodies for Alpha-1-antitrypsin (UniProt ID: P01009) for use in western blot, immunoprecipitation and flow cytometry [version 1; peer review: 1 approved]

Alpha-1-antitrypsin (A1AT), encoded by the SERPINA1 gene, is a circulating serine protease inhibitor that protects the lung from neutrophil elastase–mediated tissue damage. Mutations in SERPINA1 cause alpha-1-antitrypsin deficiency, one of the most common hereditary causes of respiratory disease, leading to early-onset emphysema and chronic obstructive pulmonary disease, as well as hepatic complications. Given the clinical significance of A1AT and its widespread use as a biomarker, there is a critical need for high-quality antibodies to ensure reliable detection and mechanistic insights. Here we systematically characterised eighteen commercial antibodies for western blot, immunoprecipitation, and flow cytometry using a standardized knockout validation approach in human Hep G2 cells, comparing readouts in a SERPINA1 knockout line with its isogenic parental control. This work is part of a broader collaborative initiative aimed at addressing antibody reproducibility by characterising commercial antibodies for human proteins and openly sharing the results with the community. While antibody use and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific experimental needs.