MAP-tau quantification in cell lysates
This HTRF kit enables the cell-based detection of phosphorylated TAU at Serine 404, as a marker of neurodegenerative diseases.
The HTRF Phospho-TAU (Ser404) cell-based assay kit is ideal for quantifying endogenous phospho-TAU phosphorylated on Serine 404. 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.
The Phospho-TAU (Ser404) assay measures TAU when phosphorylated at Ser404. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.
The Phospho-TAU (Ser404) 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 independently of 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.
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 (Ser404) HTRF detection reagents.
This protocol enables the cells' viability and confluence to be monitored.
Detection of Phosphorylated TAU (Ser202/Thr205) 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.
3 million Hek293 cells were seeded in a 100mm Petri dish, and incubated for 72 hours at 37 °C - 5% CO2. 10µg of plasmid TAU P301L/30µL lipofectamine 2000 were combined with 5mL of cell culture medium and incubated for 24 hours.
After culture medium removal, cells were lysed with 1.5mL of lysis buffer for 30min at RT under gentle shaking.
Soluble fractions were then collected after a 10 min centrifugation. 16 µL of soluble fraction were transferred into a 384-well sv white microplate, and 4 µL of the HTRF detection reagents were added.
The HTRF signal was recorded after an overnight incubation.
50,000 human SH-SY5Y cells were plated in 96-well plates and incubated for 72h at 37 °C - 5% CO2. Medium was removed and cells were lysed with 50µl of lysis buffer for 30min at RT under gentle shaking.
Alcaline phosphatase (25U) was added with the appropriate buffer, and removed 1 hour later. 16 µL of lysate were transferred into a 384-well sv white microplate, and 4 µL of the HTRF phospho-TAU (Ser404) detection reagents were added. The HTRF signal was recorded after an overnight incubation.
Human SH-SY5Y cells were grown in a T175 flask at 37 °C, 5% CO2 until 80% confluency. Cell culture medium was discarded and cells were lysed with 3 mL of supplemented lysis buffer for 30 min at room temperature. Soluble fractions were then collected after a 10 min centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 16 µL of each dilution were dispensed and analyzed side-by-side by Western Blot and by HTRF.
Using the HTRF phospho-TAU (Ser404) cellular assay, just 2,500 cells were sufficient for minimum signal detection, while 10,000 cells were needed for a Western Blot signal.
The HTRF cellular assays are at least 4-fold more sensitive than the Western Blot.
TAU displays 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 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.
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