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Specificity of anti-tau antibodies when analyzing mice models of Alzheimer's disease: problems and solutions.

Petry FR, Pelletier J, Bretteville A, Morin F, Calon F, Hébert SS, Whittington RA, Planel E - PLoS ONE (2014)

Bottom Line: For polyclonal anti-tau antibodies, some displayed non-specificity (pS262, pS409) while others did not (pS199, pT205, pS396, pS404, pS422, A0024).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.

View Article: PubMed Central - PubMed

Affiliation: Université Laval, Faculté de Médecine, Départment de Psychiatrie et Neurosciences, Québec, Canada; Centre de Recherche du CHU de Québec, CHUL, Axe Neurosciences, Québec, Canada.

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.

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Related in: MedlinePlus

Analysis of tau signal with polyclonal antibodies by Western blotting.Proteins were extracted from the cortex of 3 mouse lines: control mice (WT and Hypothermic), Tau KO mice and 3xTg-AD mice. Proteins were separated by SDS-PAGE and then identified with the following polyclonal antibodies: A: Total Tau, B: pS199, C: pS396, D: pS404, E: pT205, F: pS422, G: pS262 and H: pS409. Normal anti-rabbit secondary antibodies were used to detect primary antibodies. The heat stable fraction was used to remove non-specificity: I: pS262 and J: pS409. Quantifications of the blots are available in Figure S5.
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pone-0094251-g007: Analysis of tau signal with polyclonal antibodies by Western blotting.Proteins were extracted from the cortex of 3 mouse lines: control mice (WT and Hypothermic), Tau KO mice and 3xTg-AD mice. Proteins were separated by SDS-PAGE and then identified with the following polyclonal antibodies: A: Total Tau, B: pS199, C: pS396, D: pS404, E: pT205, F: pS422, G: pS262 and H: pS409. Normal anti-rabbit secondary antibodies were used to detect primary antibodies. The heat stable fraction was used to remove non-specificity: I: pS262 and J: pS409. Quantifications of the blots are available in Figure S5.

Mentions: We finally performed Western blot analysis to examine the specificity of polyclonal anti-tau antibodies (Table 1). With almost all polyclonal antibodies, the non-specific bands observed previously in TKO mice did not appear (Fig. 7A, B, C). Quantification of the blots is available in Figure S5. Moreover, for most antibodies tested, the tau signal was clearly apparent above 50 kDa in WT and 3xTg-AD mice without interference from others proteins. However, some epitopes such as pS262 and pS409 produced non-specific bands at different molecular weights (Fig. 7G, H). In an attempt to improve the immuno-reactivity of these latter antibodies, we tested them in HS fraction [16]. Here, heat treatment greatly improved pS262, but not pS409, specificity (Fig. 7I, J).


Specificity of anti-tau antibodies when analyzing mice models of Alzheimer's disease: problems and solutions.

Petry FR, Pelletier J, Bretteville A, Morin F, Calon F, Hébert SS, Whittington RA, Planel E - PLoS ONE (2014)

Analysis of tau signal with polyclonal antibodies by Western blotting.Proteins were extracted from the cortex of 3 mouse lines: control mice (WT and Hypothermic), Tau KO mice and 3xTg-AD mice. Proteins were separated by SDS-PAGE and then identified with the following polyclonal antibodies: A: Total Tau, B: pS199, C: pS396, D: pS404, E: pT205, F: pS422, G: pS262 and H: pS409. Normal anti-rabbit secondary antibodies were used to detect primary antibodies. The heat stable fraction was used to remove non-specificity: I: pS262 and J: pS409. Quantifications of the blots are available in Figure S5.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4008431&req=5

pone-0094251-g007: Analysis of tau signal with polyclonal antibodies by Western blotting.Proteins were extracted from the cortex of 3 mouse lines: control mice (WT and Hypothermic), Tau KO mice and 3xTg-AD mice. Proteins were separated by SDS-PAGE and then identified with the following polyclonal antibodies: A: Total Tau, B: pS199, C: pS396, D: pS404, E: pT205, F: pS422, G: pS262 and H: pS409. Normal anti-rabbit secondary antibodies were used to detect primary antibodies. The heat stable fraction was used to remove non-specificity: I: pS262 and J: pS409. Quantifications of the blots are available in Figure S5.
Mentions: We finally performed Western blot analysis to examine the specificity of polyclonal anti-tau antibodies (Table 1). With almost all polyclonal antibodies, the non-specific bands observed previously in TKO mice did not appear (Fig. 7A, B, C). Quantification of the blots is available in Figure S5. Moreover, for most antibodies tested, the tau signal was clearly apparent above 50 kDa in WT and 3xTg-AD mice without interference from others proteins. However, some epitopes such as pS262 and pS409 produced non-specific bands at different molecular weights (Fig. 7G, H). In an attempt to improve the immuno-reactivity of these latter antibodies, we tested them in HS fraction [16]. Here, heat treatment greatly improved pS262, but not pS409, specificity (Fig. 7I, J).

Bottom Line: For polyclonal anti-tau antibodies, some displayed non-specificity (pS262, pS409) while others did not (pS199, pT205, pS396, pS404, pS422, A0024).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.

View Article: PubMed Central - PubMed

Affiliation: Université Laval, Faculté de Médecine, Départment de Psychiatrie et Neurosciences, Québec, Canada; Centre de Recherche du CHU de Québec, CHUL, Axe Neurosciences, Québec, Canada.

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.

Show MeSH
Related in: MedlinePlus