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 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.
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
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Get the brochure about technology comparison. - Brochures
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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
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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
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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
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
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