Abstract
The transient receptor potential cation channel family member ankyrin 1 (TRPA1) is a potential target for several diseases, but detection of human TRPA1 (hTRPA1) protein in cells and tissues is problematic as rigorous antibody validation is lacking. We expressed hTRPA1 in a TRPA1-negative cell line to evaluate 5 commercially available antibodies by western blotting, immunofluorescence, immunocytochemistry and flow cytometry. The three most cited anti-TRPA1 antibodies lacked sensitivity and/or specificity, but two mouse monoclonal anti-TRPA1 antibodies detected hTRPA1 specifically in the above assays. This enabled the development of a flow cytometry assay, which demonstrated strong expression of TRPA1 in human lung myofibroblasts, human airway smooth muscle cells but not lung mast cells. The most cited anti-TRPA1 antibodies lack sensitivity and/or specificity for hTRPA1. We have identified two anti-TRPA1 antibodies which detect hTRPA1 specifically. Previously published data regarding human TRPA1 protein expression may need revisiting.
Highlights
The transient receptor potential cation channel family member ankyrin 1 (TRPA1) is a potential target for several diseases, but detection of human TRPA1 protein in cells and tissues is problematic as rigorous antibody validation is lacking
The transient receptor potential cation channel family member ankyrin 1 (TRPA1) is an ion channel with high Ca2+ permeability that is activated by numerous noxious stimuli and by multiple products of oxidative stress[1,2,3,4]
Much of our knowledge about expression of TRPA1 protein in human cells and tissue has relied on antibodies where validation of specificity was not undertaken or not presented
Summary
The transient receptor potential cation channel family member ankyrin 1 (TRPA1) is a potential target for several diseases, but detection of human TRPA1 (hTRPA1) protein in cells and tissues is problematic as rigorous antibody validation is lacking. In order to validate antibodies, we generated positive and negative controls using a dual promoter vector to co-express hTRPA1 with a GFP reporter in a cell line that does not contain detectable levels of endogenous hTRPA1. We used these cells to evaluate the 3 most commonly used anti-TRPA1 antibodies according to the antibody database CiteAb17; all of these are polyclonal rabbit (Table 1).
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