Phospho-BAD (Ser112) cellular kit
Highly adaptable assay to quantify phospho-BAD (Ser112)
Phospho-GSK3 beta (Ser9) cellular kit HTRF®
The phospho-GSK3 beta (Ser9) kit is designed for the cell-based detection of Serine 9 phosphorylation on GSK3 beta.
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Ease-of-use
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Highly specific
Overview
The Phospho-GSK3ß (Ser9) assay kits are designed for the robust quantification of GSK3ß modulation when phosphorylated on Ser9 as an ubiquitous readout of major pathways. GSK3 is involved in PI3K/AKT signaling, Wnt/ß-Catenin, Hedgehog, Notch, glycogen synthesis, and cytoskeleton polymerization, which control protein synthesis, cell proliferation, migration, inflammation, immune response, glucose regulation, and apoptosis.
Benefits
- SPECTIFICITY
- SPECIFICITY
Phospho-GSK3ß (Ser9) assay principle
The Phospho-GSK3ß (Ser9) assay measures GSK3ß when phosphorylated at Ser9. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-GSK3ß (Ser9) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent 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 proteins phosphorylation state under a no-wash assay format.
Phospho-GSK3ß (Ser9) 2-plate assay protocol
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 adding Phospho-GSK3ß (Ser9) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-GSK3ß (Ser9) 1-plate assay protocol
Detection of Phosphorylated GSK3ß (Ser9) 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.
HTRF assay compared to Western Blot using phospho-GSK3ß and total-GSK3ß cellular assays
Human MCF-7 cells were grown in a T175 flask at 37 °C, 5% CO2 until 80% confluency. After a 1.72 µM insulin stimulation for 30 min, 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. By using HTRF phospho-GSK3ß (Ser9) and total-GSK3ß cellular assays, just 1,500 cells are sufficient for minimal signal detection, while 12,500 cells are needed for a Western Blot signal. The HTRF cellular assays are at least 4-fold more sensitive than the Western Blot.
Inhibition effect of UCN-01 on the phosphorylation of GSK3 using phospho-GSK3ß assay
100,000 human MCF-7 cells were plated in 96 well plates and incubated for 24h at 37 °C - 5% CO2. After incubation with increasing concentrations of UCN-01 (2h), cells were stimulated with insulin at 1.72µM (30min). Medium was then 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-GSK3ß (Ser9) detection reagents were added. The HTRF signal was recorded after an overnight incubation.
Insulin dose-response on MCF-7 cells using phospho-GSK3ß (Ser9) cellular assay
100,000 human MCF-7 cells were plated in 96 well plates and incubated for 24h at 37 °C - 5% CO2. After incubation with increasing concentrations of Insulin (30min), 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-GSK3ß (Ser9) detection reagents were added. The HTRF signal was recorded after an overnight incubation.
Compatibility of the HRTF phospho-GSK3ß (Ser9) cellular assays with different cell lines
100,000 or 200,000 cells of different cell lines were plated in 96 well plates and incubated for 24h at 37 °C - 5% CO2. After incubation with or without UCN-01 at 1µg/ml (2h), cells were stimulated with 1.72µM of insulin (30min). 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-GSK3ß (Ser9) detection reagents were added. The HTRF signal was recorded after an overnight incubation.
GSK3 Simplified Pathway
GSK3 is active in numerous central intracellular signaling pathways and regulates important cellular processes from proliferation and immune responses, to glucose regulation and apoptosis. GSK3 kinase phosphorylation inhibits the activity of downstream targets. GSK3 is also regulated by cellular localization and by protein complex formation. In the insulin pathway, insulin activates PI3K, which phosphorylates AKT. In turn, activated AKT phosphorylates GSK3a on Ser21 and GSK3ß on Ser9, resulting in GSK-3 inactivation and inducing activation of glycogen synthase by dephosphorylation. Dephosphorylated GSK3 inactivates glycogen synthase, which converts glucose to glycogen. Thus, GSK3 is considered a versatile potential target for various disease research applications.
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling
Analyse of PI3K/AKT/mTor translational control pathway - Application Notes
Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers
HTRF cellular phospho-protein assays
Physiologically relevant results fo fast flowing research - Flyers
Biomarker and Cell Signaling Assays for Fibrosis and NASH
HTRF and Alpha solutions for NASH - Flyers
Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers
HTRF assays for Oncology and Inflammation
Signaling in the Immune System - Brochures
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
New cell-based HTRF® assays for the exploration of Wnt signaling pathway
Investigation of the endogenous AKT/GSK3 and Wnt pathways - 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
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences
Insider Tips for successful sample treatment - Technical Notes
HTRF Alpha-tubulin Housekeeping kit
Properly interpret your compound effect - Application Notes
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
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
A single technology for 2D cells, 3D cells, and xenograft models - Posters
Best practices for analyzing tumor xenografts with HTRF phospho assays
Protocol for tumor xenograft analysis with HTRF - Technical Notes
How to run a cell based phospho HTRF assay
What to expect at the bench - Videos
Unleash the potential of your phosphorylation research with HTRF
Unmatched ease of use, sensitivity and specificity assays - Videos
Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening
In collaboration with Bayer - Scientific Presentations
Product Insert GSK3 b P-S9 Kit / 64GPBPEG-64GPBPEH
64GPBPEG-64GPBPEH - Product Insert
All your HTRF assays in one document! - Catalog
A guide to Homogeneous Time Resolved Fluorescence
General principles of HTRF - Guides
How HTRF compares to Western Blot and ELISA
Get the brochure about technology comparison. - Brochures
Unleash the potential of your phosphorylation research with HTRF
A fun video introducing you to phosphorylation assays with HTRF - Videos
How to run a cell based phospho HTRF assay
3' video to set up your Phospho assay - Videos
Molecular basis of neuroinflammation and neurodegeneration diseases
The essential guide for extending your knowledge on the molecular mechanisms of neurodegenerative diseases - Guides
Neurodegeneration and its main related diseases
Discover this infographic design on neurodegenerative diseases - Infographics
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
Safety Data Sheet GSK3 b P-S9 Kit / 64GPBPEG
64GPBPEG - Safety Data Sheet
Safety Data Sheet (DEU) GSK3 b P-S9 Kit / 64GPBPEG
64GPBPEG - Safety Data Sheet
Safety Data Sheet (ELL) GSK3 b P-S9 Kit / 64GPBPEG
64GPBPEG - Safety Data Sheet
Safety Data Sheet (FRA-FR) GSK3 b P-S9 Kit / 64GPBPEG
64GPBPEG - Safety Data Sheet
Safety Data Sheet (ITA) GSK3 b P-S9 Kit / 64GPBPEG
64GPBPEG - Safety Data Sheet
Safety Data Sheet (SPA) GSK3 b P-S9 Kit / 64GPBPEG
64GPBPEG - Safety Data Sheet
Safety Data Sheet (ENG-GB) GSK3 b P-S9 Kit / 64GPBPEG
64GPBPEG - Safety Data Sheet
Safety Data Sheet (ENG-US) GSK3 b P-S9 Kit / 64GPBPEG
64GPBPEG - Safety Data Sheet
Safety Data Sheet GSK3 b P-S9 Kit / 64GPBPEH
64GPBPEH - Safety Data Sheet
Safety Data Sheet (DEU) GSK3 b P-S9 Kit / 64GPBPEH
64GPBPEH - Safety Data Sheet
Safety Data Sheet (ELL) GSK3 b P-S9 Kit / 64GPBPEH
64GPBPEH - Safety Data Sheet
Safety Data Sheet (FRA-FR) GSK3 b P-S9 Kit / 64GPBPEH
64GPBPEH - Safety Data Sheet
Safety Data Sheet (ITA) GSK3 b P-S9 Kit / 64GPBPEH
64GPBPEH - Safety Data Sheet
Safety Data Sheet (SPA) GSK3 b P-S9 Kit / 64GPBPEH
64GPBPEH - Safety Data Sheet
Safety Data Sheet (ENG-GB) GSK3 b P-S9 Kit / 64GPBPEH
64GPBPEH - Safety Data Sheet
Safety Data Sheet (ENG-US) GSK3 b P-S9 Kit / 64GPBPEH
64GPBPEH - Safety Data Sheet
Plate Reader Requirement
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