Commercially available antibodies directed against alpha-adrenergic receptor subtypes and other G protein-coupled receptors with acceptable selectivity in flow cytometry experiments Journal Article

Local Library Link: Find It @ Loyola

Authors: Tripathi, A; Gaponenko, V.; Majetschak, M
Article Title: Commercially available antibodies directed against alpha-adrenergic receptor subtypes and other G protein-coupled receptors with acceptable selectivity in flow cytometry experiments
Abstract: Several previous reports suggested that many commercially available antibodies directed against G protein-coupled receptors (GPCR) lack sufficient selectivity. Accordingly, it has been proposed that receptor antibodies should be validated by at least one of several criteria, such as testing tissues or cells after knockout or silencing of the corresponding gene. Here, we tested whether 12 commercially available antibodies directed against alpha-adrenergic receptor (AR) subtypes (alpha1A/B/D, alpha2A/B/C), atypical chemokine receptor 3 (ACKR3), and vasopressin receptor 1A (AVPR1A) suffice these criteria. We detected in flow cytometry experiments with human vascular smooth muscle cells that the fluorescence signals from each of these antibodies were reduced by 46 +/- 10 %-91 +/- 2 % in cells treated with commercially available small interfering RNA (siRNA) specific for each receptor, as compared with cells that were incubated with non-targeting siRNA. The tested antibodies included anti-ACKR3 (R Systems, mab42273), for which specificity has previously been demonstrated. Staining with this antibody resulted in 72 +/- 5 % reduction of the fluorescence signal after ACKR3 siRNA treatment. Furthermore, staining with anti-alpha1A-AR (Santa Cruz, sc1477) and anti-ACKR3 (Abcam, ab38089), which have previously been reported to be non-specific, resulted in 70 +/- 19 % and 80 +/- 4 % loss of the fluorescence signal after alpha1A-AR and ACKR3 siRNA treatment, respectively. Our findings demonstrate that the tested antibodies show reasonable selectivity for their receptor target under our experimental conditions. Furthermore, our observations suggest that the selectivity of GPCR antibodies depends on the method for which the antibody is employed, the species from which cells/tissues are obtained, and on the type of specimens (cell, tissue/cell homogenate, or section) tested.
Journal Title: Naunyn-Schmiedeberg's archives of pharmacology
Volume: 389
Issue: 2
ISSN: 1432-1912; 0028-1298
Publisher: Unknown  
Journal Place: Germany
Date Published: 2016
Start Page: 243
End Page: 248
Language: eng
DOI/URL:

10.1007/s00210-015-1196-0
Notes: LR: 20160120; GR: R01 GM107495/GM/NIGMS NIH HHS/United States; GR: T32 GM008750/GM/NIGMS NIH HHS/United States; JID: 0326264; NIHMS744737; OID: NLM: NIHMS744737 [Available on 02/01/17]; OID: NLM: PMC4718873 [Available on 02/01/17]; OTO: NOTNLM; PMCR: 2017/02/01 00:00; 2015/11/02 [received]; 2015/12/02 [accepted]; 2015/12/14 [aheadofprint]; ppublish