MAP-tau quantification in cell lysates
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Physiologically relevant results fo fast flowing research - Flyers
Insider Tips for successful sample treatment - Technical Notes
HTRF and WB compatible guidelines - Technical Notes
Protocol for tumor xenograft analysis with HTRF - Technical Notes
Mastering the art of cell signaling assays optimization - Guides
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
A solution for phospho-protein analysis in metabolic disorders - Posters
Detailed protocol and direct comparison with WB - Posters
A single technology for 2D cells, 3D cells, and xenograft models - Posters
PI3K/AKT/mTor translational control pathway - Posters
Analysis of a large panel of diverse biological samples and cellular models - Posters
One technology across all samples - Application Notes
Tumor xenograft analysis: HTRF versus Western blot - Application Notes
Valuable guidelines for efficiently analyzing and interpreting results - Application Notes
Increased flexibility of phospho-assays - Application Notes
Analyse of PI3K/AKT/mTor translational control pathway - Application Notes
In collaboration with Bayer - Scientific Presentations
A fun video introducing you to phosphorylation assays with HTRF - Videos
Get the brochure about technology comparison. - Brochures
New insight into neuroinflammation research - Videos
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Phospho-TAU (Ser202/Thr205) cellular kit
Simple, all-in-one kit for robust detection of Phospho-TAU S202/T205
Phospho-Tau (Ser422) cellular kit
Simple, all-in-one HTRF kit for robust detection of Phospho-TAU S422
Phospho-Tau (Ser356) cellular kit
Simple, all-in-one HTRF kit for robust detection of Phospho-TAU S356