Phospho-TAU (Thr181) assay principle

The Phospho-TAU (Thr181) assay measures TAU when phosphorylated at Thr181. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.

The Phospho-TAU (Thr181) assay uses 2 labeled antibodies: one with a donor fluorophore, and the other with an acceptor. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, and the second for its ability to recognize the protein independent from its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies, and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein’s phosphorylation state under a no-wash assay format.

Phospho-TAU (Thr181) 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a 384-well low volume detection plate before the addition of Phospho-TAU (Thr181) HTRF detection reagents.

This protocol enables the cells' viability and confluence to be monitored.

Phospho-TAU (Thr181) 1-plate assay protocol

Detection of Phosphorylated TAU (Thr181) with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required.

This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

Assessment of Tau T181 phosphorylation after BIO-induced GSK3 inhibition in human SH-SY5Y cell line

Human SH-SY5Y cells were plated at 100,000 cells/well in a 96-well plate. After 24 h incubation at 37 °C, 5% CO2, the cells were treated with increasing concentrations of GSK3α/β inhibitor BIO (6-bromoindirubin-3-oxime) for 3h. Then the medium was removed and 50 µL of supplemented lysis buffer 1X were added. After 30min lysis at RT under gentle shaking, 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho-Tau (Thr181) or total Tau detection reagents were added. The HTRF signal was recorded after an overnight incubation.

BIO-induced GSK3α/β inhibition leads to a weak inhibition of Tau phosphorylation on Threonine 181, whereas the Tau expression level remains stable in the same experimental conditions. This result is consistent with the role of different kinases such as GSK3, p38MAPK, ERK, or JNK, known to be involved in the phosphorylation of Tau on residue T181.

Assessment of Tau T181 phosphorylation after BIO-induced GSK3 inhibition in mouse Neuro2a cell line

Mouse Neuro2a cells were plated at 100,000 cells/well in a 96-well plate. After 24 h incubation at 37 °C, 5% CO2, the cells were treated with increasing concentrations of GSK3α/β inhibitor BIO for 3h. Then the medium was removed and 50 µL of supplemented lysis buffer 1X were added. After 30min lysis at RT under gentle shaking, 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho-Tau (Thr181) or total Tau detection reagents were added. The HTRF signal was recorded after an overnight incubation.

In mouse Neuro2a cells, BIO-induced GSK3α/β inhibition leads to a weak inhibition of Tau phosphorylation on Threonine 181. Note that the total Tau assay is human specific and does not cross react with mouse models.

HTRF phospho-Tau (Thr181) assay compared to Western Blot

Human Neuroblastoma SH-SY5Y cells were seeded in a T175 flask in complete culture medium and incubated for 2 days at 37°C, 5% CO2. Then the cells were lysed with 3 mL of supplemented lysis buffer#1 for 30min at RT under gentle shaking. Soluble supernatants were collected after a 10 minute centrifugation.

Serial dilutions of the cell lysate were performed in the supplemented lysis buffer, and 16 µL of each dilution were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF phospho-Tau (Thr181) detection reagents. Equal amounts of lysates were used for a side by side comparison between Western Blot and HTRF.

This result demonstrates that the phospho-Tau (Thr181) assay is 4-fold more sensitive than the Western Blot, at least under these experimental conditions.