Phospho-GSK3 beta (Ser9) cellular kit
Simple, all-in-one kit for robust detection of Total GSK3?
This HTRF kit enables the cell-based detection of phosphorylated TAU at Threonine 181, as a marker of neurodegenerative diseases.
The HTRF Phospho-TAU (Thr181) cell-based assay kit is ideal for quantifying endogenous phospho-TAU phosphorylated on Threonine 181. 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 (Thr181) assay measures TAU when phosphorylated at Thr181. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.
The Phospho-TAU (Thr181) 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.
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 (Thr181) HTRF detection reagents.
This protocol enables the cells' viability and confluence to be monitored.
Detection of Phosphorylated TAU (Thr181) 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 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 3h. 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 (Thr181) or total Tau detection reagents were added. The HTRF signal was recorded after an overnight incubation.
BIO-induced GSK3α/β inhibition leads to a weak inhibition of Tau phosphorylation on Threonine 181, whereas the Tau expression level remains stable in the same experimental conditions. This result is consistent with the role of different kinases such as GSK3, p38MAPK, ERK, or JNK, known to be involved in the phosphorylation of Tau on residue T181.
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 for 3h. 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 (Thr181) or total Tau detection reagents were added. The HTRF signal was recorded after an overnight incubation.
In mouse Neuro2a cells, BIO-induced GSK3α/β inhibition leads to a weak inhibition of Tau phosphorylation on Threonine 181. Note that the total Tau assay is human specific and does not cross react with mouse models.
Brain samples obtained from Amyloid Beta treated mice kindly provided by Amylgen (Link to your website to be defined) and 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 each brain extract 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 of 4 µL of the HTRF phospho-Tau (Thr181) or total Tau detection reagents. The HTRF signal was recorded after an overnight incubation.
The graphs show that HTRF Phospho-Tau (Thr181) enables the detection of Tau phosphorylated on Threonine 181 in both human and mouse samples obtained from brain tissues, whereas HTRF Total Tau kit is human specific.
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 phospho-Tau (Thr181) 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 (Thr181) assay is 4-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.
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Valuable guidelines for efficiently analyzing and interpreting results - Application Notes
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In collaboration with Bayer - Scientific Presentations
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