This HTRF kit can be combined with our phospho-Tau kits. The kit is able to detect phosphorylated and unphosphorylated Tau protein in the same way.

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  • All inclusive kit All inclusive kit
  • High sensitivity High sensitivity
  • Faster and more convenient than ELISA Faster and more convenient than ELISA

This HTRF kit can be combined with our phospho-Tau kits. The kit is able to detect phosphorylated and unphosphorylated Tau protein in the same way.

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Overview

The Total Tau cell-based assay is designed to monitor the expression level of Tau proteins, both phosphorylated and unphosphorylated. It uses the same buffers as our Phospho-Tau kits, and enables the analysis of phosphorylated and total proteins from a single sample.

TAU (Tubulin Associated Units), also called MAPT (Microtubule Associated Protein Tau), is a phosphoprotein largely expressed in neurons of the Central Nervous System and involved in microtubule assembly and stability.

Tau contains around of 85 potential phosphosites that can be largely phosphorylated by many kinases such as CDK5, MAP kinase, or GSK3β, the main kinase of Tau. Tau hyperphosphorylation induces a microtubule disruption, Tau aggregation into paired helical filaments or neurofibrillary tangles, and cell death.

Tau hyperphosphorylation and aggregation are involved in numerous neurodegenerative diseases such as Tauopathy, and Parkinson's and Alzheimer's Diseases. These two phenomena associated with amyloid-β aggregation are major hallmarks of Alzheimer's Disease.

Benefits

  • STUDY OF ALZHEIMER'S DISEASE
  • DISCOVER PROTAC DRUGS
  • NORMALIZATION OF PHOSPHO TAU DETECTION

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 assay principle

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 2-plate assay protocol

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.


Total Tau  1-plate assay protocol

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.

Total Tau at different cell densities in SH-SY5Y +/- BIO

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.

BIO dose-response on 100,000 SH-SY5Y cells/ well

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.

Detection of phospho-Tau (T181) and Total Tau in human AD brain extracts

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.

Western Blot and HTRF sensitivity comparison on SH-SY5Y control lysate

Role of the protein Tau in Alzheimer's Disease pathway

Tau has a prominent role in the pathogenesis of Alzheimer's Disease. It becomes hyperphosphorylated and aggregates, forming filaments, which can further condense into neurofibrillary tangles. Tau aggregates may propagate pathology by spreading from cell to cell in a prion-like manner. Drugs modulating Tau hyperphosphorylation and reducing Tau aggregation are viable therapeutic approaches.


The physiological role of Tau protein is to promote the assembly and stability of microtubules. Six isoforms of Tau have been described, ranging from 352 to 441 residues coming from exons 2, 3, and 10, that are alternatively spliced. The longest isoform of Tau (Tau-441) contains 85 putative phosphorylation sites, half of which have been confirmed experimentally.

Simplified pathway for Tau assays

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Side-by-side comparison of HTRF, Western Blot, ELISA and AlphaScreen® SureFire®

Do all cell-based kinase assays perform similarly? - Posters

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

Product Insert Tau total kit / 64NTAUPEG-64NTAUPEH

64NTAUPEG-64NTAUPEH - Product Insert

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