Antigen retrieval by citrate solution improves western blot signal

Abstract

In the present work, we describe and evaluate an additional step to the standard western blot protocol to increase signal strength after revealing. Weak or absence of signal is a common issue in western blot protocol leading to unexpected results. In our Antigen Retrieval for Western Blot Method (ARWB method), after transfer, the membrane was incubated in a citrate buffer following normal antigen retrieval procedure used for immunohistochemistry. Later, standard protocol was performed in order to reveal and compare with unexposed membranes to this antigen retrieval step. Signal in bands obtained by the modified protocol resulted significantly higher (in all 13 antibodies analyzed) compared to standard protocol. Some bands were only visible after citrate incubation. This method is a simple and economical way to improve results in western blot analysis. • The ARWB method significantly increases band’s density in all antibodies analyzed. • Protein localization does not influence the efficacy of the ARWB method since membrane and citoplasmatic proteins bands increase their signal in a similar way after the protocol is performed. • This ARWB method is simple, safe, economical and undoubtedly helpful in immunoblotting for proteins with weak signal. Method name: Antigen Retrieval for Western Blot Technique (ARWB method), Keywords: Antigen retrieval, SDS-PAGE, Citrate buffer, Antibody/epitope interaction, Novel step