Western Blot of Tau Protein from Mouse Brains Extracts: How to Avoid Signal Artifacts - PubMed (original) (raw)

Western Blot of Tau Protein from Mouse Brains Extracts: How to Avoid Signal Artifacts

Parissa Fereydouni-Forouzandeh et al. Methods Mol Biol. 2024.

Abstract

Tau is a microtubule-associated protein enriched in the axonal compartment. Its most well-known function is to bind and stabilize microtubules. In Alzheimer's disease and other neurodegenerative diseases known as tauopathies, tau undergoes several abnormal post-translational modifications including hyperphosphorylation, conformational changes, oligomerization, and aggregation. Numerous mouse models of tauopathies have been developed, and Western blotting remains an invaluable tool in studying tau protein physiological and pathological changes in these models. However, many of the antibodies that have been developed to analyze tau post-translational modifications are mouse monoclonal, which are at risk of producing artifactual signals in Western blotting procedures. This risk does not arise due to their lack of specificity, but rather because the secondary antibodies used to detect them will also react with the heavy chain of endogenous mouse immunoglobulins (Igs), leading to a non-specific signal at the same molecular weight as tau protein (around 50 kDa). Here, we present the use of anti-light-chain secondary antibodies as a simple and efficient technique to prevent non-specific Ig signals around 50 kDa. We demonstrate the efficacy of this method by either eliminating or identifying artifactual signals when using monoclonal antibodies directed at non-phosphorylated epitopes (T49, Tau3R, Tau4R), phosphorylated epitopes (MC6, AT180, CP13), or an abnormal tau conformation (MC1), in wild-type (WT) mice with tau hyperphosphorylation (hypothermic), transgenic mice overexpressing human tau (hTau mice), and tau knockout (TKO) mice.

Keywords: Alzheimer’s disease; Antibody specificity; Light-chain antibody; Mouse models; Tau phosphorylation.

© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

PubMed Disclaimer

References

    1. Wegmann S, Biernat J, Mandelkow E (2021) A current view on Tau protein phosphorylation in Alzheimer’s disease. Curr Opin Neurobiol 69:131–138. https://doi.org/10.1016/j.conb.2021.03.003 -DOI -PubMed
    1. Avila J, Lucas JJ, Pérez M et al (2004) Role of tau protein in both physiological and pathological conditions. Physiol Rev 84(2):361–384. https://doi.org/10.1152/physrev.00024.2003 -DOI -PubMed
    1. Duff K, Planel E (2005) Untangling memory deficits. Nat Med 11(8):826–827. https://doi.org/10.1038/nm0805-826 -DOI -PubMed
    1. Bretteville A, Planel E (2008) Tau aggregates: toxic, inert, or protective species? J Alzheimers Dis 14(4):431–436. https://doi.org/10.3233/JAD-2008-14411
    1. Bass JJ, Wilkinson DJ, Rankin D et al (2017) An overview of technical considerations for Western blotting applications to physiological research. Scand J Med Sci Sports 27(1):4–25. https://doi.org/10.1111/sms.12702 -DOI -PubMed

MeSH terms

Substances

LinkOut - more resources