Tau Aggregation Kit
Tau aggregation detection in cell and brain extracts
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.
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® phospho-protein platform facilitates the dissection of signaling pathways involved in insulin resistance and metabolic disorders
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
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
Implement HTRF phospho-protein assays at every step of the drug discovery process, from in vitro to in vivo models
A single technology for 2D cells, 3D cells, and xenograft models - Posters
Simplify complex pathway dissection by combining the power of HTRF® cellular phospho-assays and the flexibility of the CyBi®-FeliX liquid handling system
PI3K/AKT/mTor translational control pathway - 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
From 2D, 3D cell cultures to xenografts: A smart HTRF platform to maximize anticancer drug discovery
One technology across all samples - Application Notes
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
Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening
In collaboration with Bayer - Scientific Presentations
Unleash the potential of your phosphorylation research with HTRF
A fun video introducing you to phosphorylation assays with HTRF - Videos
How HTRF compares to Western Blot and ELISA
Get the brochure about technology comparison. - Brochures
On-demand webinar: Linking Neuroinflammation and Neurodegeneration
New insight into neuroinflammation research - Videos
Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection
Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes
Assessment of drug efficacy and toxicity by combining innovative technologies
Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes
Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)
Learn how to reduce time and sample consumption - Application Notes
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.Let's find your reader
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