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 Ser202/Thr205, as a marker of neurodegenerative diseases.
The HTRF Phospho-TAU (Ser202/Thr205) cell-based assay kit is ideal for quantifying endogenous phospho-TAU phosphorylated on Serine 202/Threonine 205.
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 (Ser202/Thr205) assay measures TAU when phosphorylated at Ser202/Thr205. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.
The Phospho-TAU (Ser202/Thr205) assay uses 2 labeled antibodies: one with a donor fluorophore, the other with an acceptor. The first antibody is 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 additio,n of Phospho-TAU (Ser202/Thr205) 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.
50,000 human SH-SY5Y cells were plated in 96-well plates and incubated for 72h at 37 °C - 5% CO2.
After incubation with increasing concentrations of GSK3 inhibitors (CHIR99021 and BIO) for 3 hours, medium was removed and cells were lysed with 50µl of lysis buffer for 30min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well sv white microplate, and 4 µL of the HTRF phospho-TAU (Ser202/Thr205) 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 (Ser202/Thr205) 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. The SH-SY5Y cells were then treated with the GSK3 inhibitor, BIO, for 3 hours. After cell culture medium removal, cells were lysed with 50µl of lysis buffer for 30min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well sv white microplate, and 4 µL of the HTRF phospho-TAU (Ser202/Thr205) and α-tubulin 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 then 16 µL of each dilution were dispensed and analyzed side-by-side by Western Blot and by HTRF.
Using the HTRF phospho-TAU (Ser202/Thr205) cellular assay, just 2,500 cells were sufficient for minimum signal detection, while 5,000 cells were needed for a Western Blot signal. The HTRF cellular assays are at least 2-fold more sensitive than the Western Blot.
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 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.
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
Exploring LRRK2 phosphorylation with HTRF® assays
LRRK2 kit, a game changer in neuroscience - Posters
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
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® 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
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
Neurodegeneration and its main related diseases
Discover this infographic design on neurodegenerative diseases - Infographics
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
You might be interested in
Total alpha Synuclein cellular kit
Simple, all-in-one kit for robust detection of total alpha Synuclein