Abstract
Aggregates of hyperphosphorylated tau protein are found in a group of diseases called tauopathies, which includes Alzheimer's disease. The causes and consequences of tau hyperphosphorylation are routinely investigated in laboratory animals. Mice are the models of choice as they are easily amenable to transgenic technology; consequently, their tau phosphorylation levels are frequently monitored by Western blotting using a panel of monoclonal/polyclonal anti-tau antibodies. Given that mouse secondary antibodies can recognize endogenous mouse immunoglobulins (Igs) and the possible lack of specificity with some polyclonal antibodies, non-specific signals are commonly observed. Here, we characterized the profiles of commonly used anti-tau antibodies in four different mouse models: non-transgenic mice, tau knock-out (TKO) mice, 3xTg-AD mice, and hypothermic mice, the latter a positive control for tau hyperphosphorylation. We identified 3 tau monoclonal antibody categories: type 1, characterized by high non-specificity (AT8, AT180, MC1, MC6, TG-3), type 2, demonstrating low non-specificity (AT270, CP13, CP27, Tau12, TG5), and type 3, with no non-specific signal (DA9, PHF-1, Tau1, Tau46). For polyclonal anti-tau antibodies, some displayed non-specificity (pS262, pS409) while others did not (pS199, pT205, pS396, pS404, pS422, A0024). With monoclonal antibodies, most of the interfering signal was due to endogenous Igs and could be eliminated by different techniques: i) using secondary antibodies designed to bind only non-denatured Igs, ii) preparation of a heat-stable fraction, iii) clearing Igs from the homogenates, and iv) using secondary antibodies that only bind the light chain of Igs. All of these techniques removed the non-specific signal; however, the first and the last methods were easier and more reliable. Overall, our study demonstrates a high risk of artefactual signal when performing Western blotting with routinely used anti-tau antibodies, and proposes several solutions to avoid non-specific results. We strongly recommend the use of negative (i.e., TKO) and positive (i.e., hypothermic) controls in all experiments.
Highlights
Alzheimer’s disease (AD) is the most common neurodegenerative disease and is characterized by a progressive loss of cognitive function, leading to dementia [1,2]
When Dynabeadscleared supernatants were incubated with AT8, the tau phosphorylation signal appeared only in hypothermic mice (Fig. 5A2). These results strongly suggest that the bands observed in wild-type mice (WT) and 3xTg-AD mice correspond to endogenous Igs, at least under these conditions
We have demonstrated that several monoclonal antibodies directed against tau or phospho-tau epitopes can display non-specificity due to the property of secondary antimouse antibodies to bind to endogenous mouse Igs
Summary
Alzheimer’s disease (AD) is the most common neurodegenerative disease and is characterized by a progressive loss of cognitive function, leading to dementia [1,2]. Tau plays a role in promoting the assembly and maintenance of microtubules through its microtubule-binding domain. The capacity of tau to bind microtubules and promote stabilization and assembly is negatively regulated by its phosphorylation, in and around the microtubule binding domain [5]. Under pathological conditions, such as AD and others tauopathies, tau becomes hyperphosphorylated, resulting in reduced affinity for microtubules and self-aggregation into abnormal filaments, leading to formation of NFTs [6]. Primary anti-tau rabbit polyclonal antibodies can recognize other proteins, with similar molecular weights to that of tau, leading to non-specific bands masking or interfering with the tau signal
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