Quantifying Tau Phosphorylation Patterns

Cell lysates were resolved via SDS-PAGE (on 10% gels for tau-T40 and 15% gels for tau-K18), transferred to nitrocellulose membrane (Biorad), and blocked with 2% milk in 1X TBS for 30 min. Membranes were incubated with primary antibody overnight at 4 °C, followed by a 1 h room temperature incubation with HRP-conjugated anti-mouse or anti-rabbit antibodies (1:1000). The following primary antibodies were used: pSer202/pThr205-tau (AT8, 1:1000, Thermo Fisher Scientific, MN1020), pSer262-tau (p-S262, 1:1000, Thermo Fisher Scientific, 44-750G), pThr231-tau (AT180, 1:1000, Thermo Fisher Scientific, MN1040), pThr181-tau (AT270, 1:1000, Thermo Fisher Scientific, MN1050), tau-1 (1:1000, Millipore, MAB3420), pSer396-tau (p-S396, Thermo Fisher Scientific, 44-752G), anti-acetylated K280-tau4 (link) (ac-K280, 1:1000), tau K9JA (total tau, 1:5000, Dako, A0024), tau-T46 (1:1000, Thermo Fisher Scientific, 13-6400), and Hsc70 (1:1000, Enzo Life Sciences, SPA-815). ImageJ software (NIH) was used to quantify band intensity.

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Trzeciakiewicz H., Tseng J.H., Wander C.M., Madden V., Tripathy A., Yuan C.X, & Cohen T.J. (2017). A Dual Pathogenic Mechanism Links Tau Acetylation to Sporadic Tauopathy. Scientific Reports, 7, 44102.

Publication 2017

nitrocellulose membrane p-S396

Anti antibodies Antibodies Antibody Cell Gels Membranes Milk Mouse Nitrocellulose Rabbit Sds page Tau 1

Corresponding Organization : University of North Carolina at Chapel Hill

Other organizations : Alexion Pharmaceuticals (United States)

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Variable analysis

independent variables

Primary antibodies used (pSer202/pThr205-tau, pSer262-tau, pThr231-tau, pThr181-tau, tau-1, pSer396-tau, ac-K280, tau K9JA, tau-T46)

dependent variables

Protein expression levels

control variables

Cell lysates resolved via SDS-PAGE
Proteins transferred to nitrocellulose membrane
Membranes blocked with 2% milk in 1X TBS for 30 min
Membranes incubated with primary antibodies overnight at 4 °C
Membranes incubated with HRP-conjugated secondary antibodies (1:1000) for 1 h at room temperature
Hsc70 used as a loading control

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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