Western blot analysis of BK channel beta 1-subunit expression should be interpreted cautiously when using commercially available antibodies

Large conductance Ca2+-activated K+ (BK) channels consist of pore-forming alpha-and accessory beta-subunits. There are four beta-subunit subtypes (beta 1-beta 4), BK beta 1-subunit is specific for smooth muscle cells (SMC). Reduced BK beta 1-subunit expression is associated with SMC dysfunction in animal models of human disease, because downregulation of BK beta 1-subunit reduces channel activity and increases SMC contractility. Several anti-BK beta 1-subunit antibodies are commercially available; however, the specificity of most antibodies has not been tested or confirmed in the tissues from BK beta 1-subunit knockout (KO) mice. In this study, we tested the specificity and sensitivity of six commercially available antibodies from five manufacturers. We performed western blot analysis on BK beta 1-subunit enriched tissues (mesenteric arteries and colons) and non-SM tissue (cortex of kidney) from wild-type (WT) and BK beta 1-KO mice. We found that antibodies either detected protein bands of the appropriate molecular weight in tissues from both WT and BK beta 1-KO mice or failed to detect protein bands at the appropriate molecular weight in tissues from WT mice, suggesting that these antibodies may lack specificity for the BK beta 1subunit. The absence of BK beta 1-subunit mRNA expression in arteries, colons, and kidneys from BK beta 1-KO mice was confirmed by RT-PCR analysis. We conclude that these commercially available antibodies might not be reliable tools for studying BK beta 1-subunit expression in murine tissues under the denaturing conditions that we have used. Data obtained using commercially available antibodies should be interpreted cautiously. Our studies underscore the importance of proper negative controls in western blot analyses.