Abstract 5645: Critical variables to reduce Western blot variability

Abstract

Abstract Reliable immunodetection of proteins is a critical step in the discovery of new biomarkers and diagnostic. The immunodetection protocol consists of multiple steps including blocking of nonspecific binding sites, incubation using primary and secondary antibodies and extensive washing between steps that easily introduce bias and errors. The quality and reproducibility of results depends on multiple subjective and objective factors such as the qualification and technical skills of the personnel performing the assay and the accuracy of temporal and temperature control, especially during the immunodetection step. BlotCycler™, automated western blot processor, use fluidic control system that allows to eliminate the variability associated with immunodetection and to achieve higher sensitivity by optimized washing procedure. We used BlotCycler to analyze critical source of errors during immunodetection. Relatively small changes in duration and temperature of incubation significantly affect not only the intensity of signal but also the specificity of antibody interaction with antigen. Automated immunodetection at 4ºC significantly improve the specificity of detection and sensitivity that allows reproducible detection of low expressing proteins. Automated processing using BlotCycler allows optimization and control of critical variables leading to improved reproducibility and specificity of immunodetection and should be routinely used for new biomarker discovery and diagnostics, reliable protein quantification, primary antibody specificity testing, and optimization of primary and secondary antibodies concentrations. Citation Format: Russ Yukhananov, Alex Margulis. Critical variables to reduce Western blot variability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5645.