Don't trust the datasheet: a validation of commercial G alpha i/o primary antibodies for Western blotting
Authors: Ebert Kevin , Buckley Rebecca , Kim Sunyoung , Kapusta Daniel R. 
Published: June 2021

Primary antibodies are an integral part of Western blotting but validation of the specificity and sensitivity of antibodies by suppliers is extremely lacking. Multiple examples of this are evident in the G alpha i/o family of proteins. G alpha proteins are important signal transducing molecules for both endogenous cellular function and drug action that are divided into four families: Gs, Gi/o, Gq/11, and G12/13. These four families of proteins interact with the nearly 1000 G protein coupled receptors (GPCRs) in the human body, the largest and most diverse group of receptors. Because of the diversity of these receptors, around one third of all drugs on the market target GPCRs, which, in part, utilize different G alpha subunit proteins to evoke their downstream signaling and physiological responses. Therefore, it is important that researchers have tools; e.g., selective antibodies, that can clearly define how G alpha proteins produce their effects associated with a receptor and its ligand. Multiple commercially available G alpha protein antibodies have been employed for relative G protein quantification, but they have not been adequately verified for both their specificity and sensitivity. In this study, BL‐21 DE3 RIL competent cells were used to express and purify five protein members of the Gi/o family of G alpha proteins. Gi1, Gi2, Gi3, Go, and Gz proteins were purified using an AKTA FPLC system and a histidine trap purification column. Western blots using these proteins and multiple Gi/o primary antibodies from different vendors were used to determine the specificity of the antibodies for their advertised protein. Our results showed antibodies that were successful in binding specifically to their target and antibodies that either did not bind to the protein of interest or expressed binding to multiple members of the Gi/o family despite claims that it was selective in targeting a single protein. After determination of specificity, purified proteins were used in a 1:2 dilution series to determine the linear range of detection for each antibody that was deemed specific for a single protein. This linear range was then used for determination of sensitivity and absolute quantitation of Gi/o proteins in Sprague‐Dawley rat brain tissue, specifically punches from the paraventricular nucleus and cortex. Together, the results of these studies indicate that certain antibodies, which are available from commercial vendors for use in Western blot assays, may not be selective for a specific Gi/o subunit protein as advertised. Thus, it is essential that investigators be sure to verify that their antibody is selective and specific for their target protein in their own hands, to ensure that the use of the antibody in subsequent studies is valid.Support or Funding InformationNIH 5P30GM106392 to DK and R01GM097350 to SKThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.