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

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

Methods in molecular biology (Clifton, N.J.) (2024-03-21)
Parissa Fereydouni-Forouzandeh, Geoffrey Canet, Sofia Diego-Diàz, Emma Rocaboy, Serena Petry, Robert A Whittington, Emmanuel Planel
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Monoclonal Anti-β-Actin antibody produced in mouse, clone AC-74, purified immunoglobulin, buffered aqueous solution
Sigma-Aldrich
Anti-Tau Antibody, clone T49 (Not human), clone T49, from mouse
Sigma-Aldrich
Goat Anti-Mouse light chain Antibody, HRP conjugate, Chemicon®, from goat
Sigma-Aldrich
Anti-Tau (4-repeat isoform RD4) Antibody, clone 1E1/A6, culture supernatant, clone 1E1/A6, Upstate®
Sigma-Aldrich
Anti-Tau (3-repeat isoform RD3) Antibody, clone 8E6/C11, culture supernatant, clone 8E6/C11, Upstate®