title: Western blot analysis of BK channel β1-subunit expression should be interpreted cautiously when using commercially available antibodies.
abstract: Large conductance Ca(2+)-activated K(+) (BK) channels consist of pore-forming α- and accessory β-subunits. There are four β-subunit subtypes (β1-β4), BK β1-subunit is specific for smooth muscle cells (SMC). Reduced BK β1-subunit expression is associated with SMC dysfunction in animal models of human disease, because downregulation of BK β1-subunit reduces channel activity and increases SMC contractility. Several anti-BK β1-subunit antibodies are commercially available; however, the specificity of most antibodies has not been tested or confirmed in the tissues from BK β1-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 β1-subunit enriched tissues (mesenteric arteries and colons) and non-SM tissue (cortex of kidney) from wild-type (WT) and BK β1-KO mice. We found that antibodies either detected protein bands of the appropriate molecular weight in tissues from both WT and BK β1-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 β1-subunit. The absence of BK β1-subunit mRNA expression in arteries, colons, and kidneys from BK β1-KO mice was confirmed by RT-PCR analysis. We conclude that these commercially available antibodies might not be reliable tools for studying BK β1-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.
authors.affiliation: Neuroscience Program, Michigan State University, East Lansing, Michigan.
authors.forename: Yogesh
authors.initials: Y
authors.lastname: Bhattarai
authors.affiliation: Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan.
authors.forename: Roxanne
authors.initials: R
authors.lastname: Fernandes
authors.affiliation: Department of Pediatrics and Human Development, Michigan State University, East Lansing, Michigan.
authors.forename: Mark M
authors.initials: MM
authors.lastname: Kadrofske
authors.affiliation: Department of Pediatrics and Human Development, Michigan State University, East Lansing, Michigan.
authors.forename: Lizbeth R
authors.initials: LR
authors.lastname: Lockwood
authors.affiliation: Neuroscience Program, Michigan State University, East Lansing, Michigan Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan.
authors.forename: James J
authors.initials: JJ
authors.lastname: Galligan
authors.affiliation: Neuroscience Program, Michigan State University, East Lansing, Michigan Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan.
authors.forename: Hui
authors.initials: H
authors.lastname: Xu
keywords: Anti‐BK channel β1‐subunit antibody; BK β1‐subunit expression; BK β1‐subunit knockout; sensitivity and specificity; smooth muscle cell