Phospho-TAU (Ser404) assay principle

The Phospho-TAU (Ser404) assay measures TAU when phosphorylated at Ser404. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.

The Phospho-TAU (Ser404) 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 independently of 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 (Ser404) 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 (Ser404) HTRF detection reagents.

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

Phospho-TAU (Ser404) 1-plate assay protocol

Detection of Phosphorylated TAU (Ser202/Thr205) 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.

High phosphorylation level of TAU S404 in HEK293 cells over expressing Tau P301L mutant

3 million Hek293 cells were seeded in a  100mm Petri dish, and incubated for 72 hours at 37 °C - 5% CO2. 10µg of plasmid TAU P301L/30µL lipofectamine 2000 were combined with 5mL of cell culture medium and incubated for 24 hours.

After culture medium removal, cells were lysed with 1.5mL of lysis buffer for 30min at RT under gentle shaking.

Soluble fractions were then collected after a 10 min centrifugation. 16 µL of soluble fraction were transferred into a 384-well sv white microplate, and 4 µL of the HTRF detection reagents were added.

The HTRF signal was recorded after an overnight incubation.

Phosphorylation of TAU on Ser404 is abrogated by alkaline Phosphatase treatment

50,000 human SH-SY5Y cells were plated in 96-well plates and incubated for 72h at 37 °C - 5% CO2. Medium was removed and cells were lysed with 50µl of lysis buffer for 30min at RT under gentle shaking.

Alcaline phosphatase (25U) was added with the appropriate buffer, and removed 1 hour later. 16 µL of lysate were transferred into a 384-well sv white microplate, and 4 µL of the HTRF phospho-TAU (Ser404) detection reagents were added. The HTRF signal was recorded after an overnight incubation.

HTRF vs Western Blot comparison

Human SH-SY5Y cells were grown in a T175 flask at 37 °C, 5% CO2 until 80% confluency. Cell culture medium was discarded and cells were lysed with 3 mL of supplemented lysis buffer for 30 min at room temperature. Soluble fractions were then collected after a 10 min centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 16 µL of each dilution were dispensed and analyzed side-by-side by Western Blot and by HTRF.

Using the HTRF phospho-TAU (Ser404) cellular assay, just 2,500 cells were sufficient for minimum signal detection, while 10,000 cells were needed for a Western Blot signal.

The HTRF cellular assays are at least 4-fold more sensitive than the Western Blot.