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.

Keywords: Anti‐BK channel β1‐subunit antibody; BK β1‐subunit expression; BK β1‐subunit knockout; sensitivity and specificity; smooth muscle cell.

Figure 1.

Representative western blot obtained using anti‐BK β1‐subunit antibodies in MA, colonic, or kidney tissues from WT and BK β1‐KO mice, (A) Alomone Labs (APC‐036), (B) Pierce (PA5‐28284), and (C) Abcam (Ab3587) antibodies detected a protein band at ~28 kDa or ~38 kDa in colons or MA from both mice. The bands in Abcam sets were diminished after preincubation of the primary antibody with the competing peptide. (D) Pierce (PA1‐924), (E) Santa Cruz (sc‐14749), and (F) Biorbyt (orb‐101774) antibodies did not detect any band at ~28 kDa or ~21 kDa in MA or colons from WT mice. (G) Alomone Labs (APC‐036), Pierce (PA1‐924), Abcam (Ab3587), and Biorbyt (Orb‐101774) antibodies did not detect protein band at ~28 kDa, ~38 kDa, or ~21 kDa in kidneys from WT mice. (H) Pierce (PA5‐28284) and (I) Santa Cruz (sc‐14749) antibodies detected the protein band at ~28 kDa in kidneys from both mice. β‐actin was reblotted on each membrane after anti‐BK β1‐subunit antibody was stripped. All representative blot images from kidney are in the tissue from same WT or BK β1‐KO mouse, and blotted with primary anti‐BK β1‐subunit antibody at 1:200. Arrows indicate the manufacturer's recommended molecular weight of BK β1‐subunit protein.

Figure 2.

(A) Representative amplification plots and agarose gel separation of real‐time RT‐PCR analysis of BK β1‐subunit and GAPDH in MA, colons, and kidneys from WT and BK β1‐KO mice. The expression threshold was set at 0.22, a level above background fluorescence but within the linear phase of the amplification plot. The intersection between the threshold level and the amplification plot is the Ct value, which correlates with the amount of template in the sample. Ct values over 35 are excluded, as these values approach the sensitivity limits of the Taqman assay. Amplification of real‐time RT‐PCR products was seen at 75 bp in tissues from WT animals only. Con, nontemplate control. (B) Representative western blot obtained using anti‐BK α‐subunit antibody in colonic and kidney tissues from WT and BK β1‐KO mice. Antibody detected a protein band at ~100 kDa in all tissues from WT and BK β1‐KO mice. The signals are blocked by preincubation with the antibody competing peptide (CP). Arrows indicate the manufacturer's recommended molecular weight of BK α‐subunit protein.