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Phospho-Tau (Ser356) cellular kit HTRF®

This HTRF kit enables the cell-based detection of phosphorylated TAU at Serine 356, as a marker of neurodegenerative diseases.

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  • Ready-to-use Ready-to-use
  • High sensitivity High sensitivity
  • Rapid Rapid
  • No-wash No-wash

This HTRF kit enables the cell-based detection of phosphorylated TAU at Serine 356, as a marker of neurodegenerative diseases.

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Overview

The HTRF Phospho-TAU (Ser356) cell-based assay kit is ideal for quantifying endogenous phospho-TAU phosphorylated on Serine 356. TAU exists in different states in both Alzheimer’s (AD) and Parkinson’s (PD) Diseases.

TAU hyperphosphorylation is also a marker for multiple neurodegenerative diseases and CNS disorders.

Benefits

  • SPECIFICITY
  • PRECISION

Phospho-TAU (Ser356) assay principle

The Phospho-TAU (Ser356) assay measures TAU when phosphorylated at Ser356. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.

The Phospho-TAU (Ser356) 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 (Ser356) Assay principle

Phospho-TAU (Ser356) 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 (Ser356) HTRF detection reagents.

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

Phospho-TAU (Ser356) 2-plate assay protocol

Phospho-TAU (Ser356) 1-plate assay protocol

Detection of Phosphorylated TAU (Ser356) 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.

Phospho-TAU (Ser356) 1-plate assay protocol

Assessment of Tau S356 phosphorylation after BIO-induced 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 (Ser356) 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 356, whereas the Tau expression level remains stable under the same experimental conditions.

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

Assessment of Tau S356 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 (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 (Ser356) or total Tau detection reagents were added. The HTRF signal was recorded after an overnight incubation.


As in SH-SY5Y cells, BIO-mediated GSK3α/β inhibition leads to a complete inhibition of Tau phosphorylation on Serine 356. Note that the total Tau assay is human specific and does not cross react with mouse models.

BIO dose-response on 100,000 Neuro2a cells/ well

HTRF phospho-Tau (Ser356) 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 treated with Okadaic acid (100 nM) for 2 h and Calyculin A (100 nM) for 10 min. Following these treatments, 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 HTRF phospho-Tau (Ser356) 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 (Ser356) assay is 4-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

Analysis of the effect of aggregated beta-Amyloid on cellular signaling pathways critical for memory in Alzheimer's disease

Changes in CREB and ERK phosphorylation levels after beta-amyloid treatment - Application Notes

Detection of human tau protein aggregation

For researcher working on PD or AD - Application Notes

Cisbio lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

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

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

Insider Tips for successful sample treatment - Technical Notes

Exploring Synuclein phosphorylation and aggegation with HTRF assays

Alpha Synuclein kits, perfect tools to study synucleinopathy - Posters

HTRF Product Catalog 2020 July update

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

Exploring LRRK2 phosphorylation with HTRF® assays

LRRK2 kit, a game changer in neuroscience - Posters

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

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

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

Molecular basis of neuroinflammation and neurodegeneration diseases

The essential guide for extending your knowledge on the molecular mechanisms of neurodegenerative diseases - Guides

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Product Insert Tau P-S356 Kit / 64TS356PEG-64TS356PEH

64TS356PEG-64TS356PEH - Product Insert

On-demand webinar: Linking Neuroinflammation and Neurodegeneration

New insight into neuroinflammation research - Videos

Assays for neurosciences research

Advance your research on neurodegenerative diseases - Flyers

Plate Reader Requirement

Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.

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