Mouse Anti-Tau Recombinant Antibody (clone C5.2) (CAT#: PABS-0209)

Figure 1 Western blot (upper image) for phosphorylated tau at pS396 epitope with C.5.2

Brain samples from 24 weeks old rTg4510 and non-transgenic littermate (non-tg) mice (left image) and from 4 AD and age-matched healthy control (HC) donors (right image) were fractioned into soluble (S1) and sarkosyl-insoluble fraction (P3) and analyzed by western blot (upper image) and dot blot (lower image) for phosphorylated tau at pS396 epitope with C.5.2 (1 µg/ml). AD and HC samples were pooled from 2 males and 2 females each. All four donors were Caucasian and age-matched. Individual AD and HC brains were analyzed with similar results. By western blot, phosphorylated and non-phosphorylated human 4R0N tau with the P301L mutation (tau P301L) from the rTg4510 mice is displayed at 55 kDa, hyperphosphorylated 4R0N tau P301L is mobility-shifted and displayed at 64 and 70 kDa. Phosphorylated and non-phosphorylated six human tau isoforms in AD and HC brains are displayed at 45, 55, 60 and 65 kDa, hyperphosphorylated tau isoforms are displayed at 54, 64, 69 and 74 kDa together with the typical AD smear.

Chukwu, J. E., Pedersen, J. T., Pedersen, L. Ø., Volbracht, C., Sigurdsson, E. M., & Kong, X. P. (2018). Tau antibody structure reveals a molecular switch defining a pathological conformation of the tau protein. Scientific reports, 8(1), 1-11.

Figure 2 Dot blot (lower image) for phosphorylated tau at pS396 epitope with C.5.2

Brain samples from 24 weeks old rTg4510 and non-transgenic littermate (non-tg) mice (left image) and from 4 AD and age-matched healthy control (HC) donors (right image) were fractioned into soluble (S1) and sarkosyl-insoluble fraction (P3) and analyzed by western blot (upper image) and dot blot (lower image) for phosphorylated tau at pS396 epitope with C.5.2 (1 µg/ml). AD and HC samples were pooled from 2 males and 2 females each. All four donors were Caucasian and age-matched. Individual AD and HC brains were analyzed with similar results.

Chukwu, J. E., Pedersen, J. T., Pedersen, L. Ø., Volbracht, C., Sigurdsson, E. M., & Kong, X. P. (2018). Tau antibody structure reveals a molecular switch defining a pathological conformation of the tau protein. Scientific reports, 8(1), 1-11.

Figure 3 Representative images of prefrontal cortex from an AD and an age-matched healthy control (HC) donor processed for C5.2 immunohistochemistry.

Paraffin sections of 4 μm thickness were prepared from blocks of PFA-fixed prefrontal cortex from AD donors and non-demented controls obtained from Tissue Solutions (Glasgow, UK). The sections were deparaffinised, rehydrated and subjected to antigen retrieval by boiling in 10 mM citrate buffer pH 6 for 5 min. Endogenous peroxidase was quenched for 10 min with 1% H2O2 in PBS. Sections were then incubated 20 min in blocking buffer followed by overnight incubation in a humidified chamber at 4 °C with primary antibody at 0.1 to 1 μg/mL in incubation buffer. Between each incubation step, the sections were washed 3 × 10 min in wash buffer. Subsequently, the sections were incubated at room temperature in the following solutions; 60 min with a biotinylated secondary Ab diluted 1:200 in wash buffer; 60 min in ABC; 20 min in DAB. Finally, the sections were counterstained with hematoxylin and coverslipped.

Chukwu, J. E., Pedersen, J. T., Pedersen, L. Ø., Volbracht, C., Sigurdsson, E. M., & Kong, X. P. (2018). Tau antibody structure reveals a molecular switch defining a pathological conformation of the tau protein. Scientific reports, 8(1), 1-11.

Figure 4 C5.2 IgG ELISA

Immulon 4 HBX (extra high-binding) 96-well microtiter plates (Thermo Scientific) were coated with the same peptide used for crystallization diluted to 2 μg/mL in phosphate-buffered saline (PBS) and left overnight at 4 °C. Plates were then washed three times in PBS-T (PBS containing 0.05% Tween-20) and blocked in 5% non-fat milk and 3% bovine serum albumin in PBS for 2 hours at room temperature. Plates were subsequently washed 3 times in PBS-T before C5.2 was serially diluted (8 μg/mL-0.05 ng/mL) in PBS and incubated for 1 hour at room temperature. Then, alkaline phosphatase-conjugated goat anti-mouse IgG (Southern Biotech) was diluted 1:2000 in PBS and incubated for 30 minutes at room temperature. Finally, bound IgG was analyzed by addition of p-nitrophenyl phosphate substrate (Thermo Scientific) and measurement at 405 nm on a VersaMax Microplate Reader.

Chukwu, J. E., Pedersen, J. T., Pedersen, L. Ø., Volbracht, C., Sigurdsson, E. M., & Kong, X. P. (2018). Tau antibody structure reveals a molecular switch defining a pathological conformation of the tau protein. Scientific reports, 8(1), 1-11.

Figure 5 C5.2 Fab fluorescence polarization binding curves comparing recognition of four differentially phosphorylated peptides.

C5.2 binds peptides pS396/pS404 (black) and pS396 (blue), with KD of 15.4 nM and 14.9 nM, respectively, but does not recognize peptide pS404 (green) or the non-phosphorylated (light blue) peptide. The dip in fluorescence for the pS404 and non-phosphorylated peptides around 10 nM Fab concentration is due to protein aggregation and fluorophore quenching. Both experiments confirm C5.2's phospho-specificity for pS396.

Chukwu, J. E., Pedersen, J. T., Pedersen, L. Ø., Volbracht, C., Sigurdsson, E. M., & Kong, X. P. (2018). Tau antibody structure reveals a molecular switch defining a pathological conformation of the tau protein. Scientific reports, 8(1), 1-11.