Total Tau assay principle

The Total Tau assay quantifies the expression level of Tau in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.

The Total Tau assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of Tau in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.

Total tau 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before adding Total Tau HTRF detection reagents.

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

Total Tau 1-plate assay protocol

Detection of total Tau 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.

Detection of Tau expression level in human SH-SY5Y cells at different cell densities

Human SH-SY5Y cells were plated at different cell densities in a 96-well plate. After 24 h incubation at 37 °C, 5% CO2, 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 total Tau detection reagents were added. The HTRF signal was recorded after an overnight incubation.

The high sensitivity and dynamic range of the Total Tau assay enable the detection of the protein Tau from 25,000 cells (S/N > 5) to 100,000 SH-SY5Y cells per well.

Assessment of the expression and phosphorylation of Tau after BIO-mediated GSK3 inhibition in human SH-SHY5Y 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 1 h, followed by 2 h Okadaic acid (100nM) and 10 min Calyculin A (100nM) treatments. 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 (Ser422) or total Tau detection reagents were added. The HTRF signal was recorded after an overnight incubation.

BIO-induced GSK3α/β inhibition leads to a complete inhibition of Tau phosphorylation on Serine 422, whereas the Tau expression level remains stable in the same experimental conditions.

These results demonstrate that the HTRF Total Tau assay enables the detection of protein Tau independent from its phosphorylation state.

Detection of phospho T181 and total Tau in human brain extracts from Alzheimer's patients

Brain samples obtained from human Alzheimer's patients were prepared according to the Cisbio technical note 'Optimize your HTRF® cell signaling assays on tissues'. (link to the AN)

Protein content from brain extracts was quantified using BCA assay, and adjusted to 1mg/mL protein concentration prior to serial dilutions. Then 16µL of each dilution were transferred into a low volume white microplate before the addition 4 µL of the HTRF phospho-Tau (Thr181) or total Tau detection reagents. The HTRF signal was recorded after an overnight incubation.

HTRF Phospho-Tau (Thr181) and Total kits demonstrate high performances in human brain-derived samples.

HTRF Total Tau 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 Total Tau detection reagents. Equal amounts of lysates were used for a side by side comparison between Western Blot and HTRF.

This result demonstrates that the HTRF total Tau assay is 16-fold more sensitive than the Western Blot, at least under these experimental conditions.