The beta-Catenin (Thr41/Ser37/Ser33) kit detects GSK3 beta-induced phosphorylation of beta-Catenin.
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The beta-Catenin (Thr41/Ser37/Ser33) kit detects GSK3 beta-induced phosphorylation of beta-Catenin.
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Overview

The Phospho ß-Catenin (Thr41/Ser37/Ser33) assay kit offers a robust detection of GSK3 ß-induced phosphorylation of ß-Catenin on Thr41/Ser37/Ser33 as a readout of the canonical Wnt pathway. Phosphorylation of ß-Catenin on Thr41/Ser37/Ser33 represents the inactive form of the protein degraded by the proteasome.

Benefits

  • VALIDATED ON MANY CELL TYPES
  • SPECIFICITY

Phospho ß-Catenin (Thr41/Ser37/Ser33) assay principle

The Phospho ß-Catenin (Thr41/Ser37/Ser33) assay measures Catenin when phosphorylated at Thr41/Ser37/Ser33. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho ß-Catenin (Thr41/Ser37/Ser33) 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 protein’s phosphorylation state under a no-wash assay format.
Phospho ß-Catenin (Thr41/Ser37/Ser33) assay principle

Phospho ß-Catenin (Thr41/Ser37/Ser33) 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 ß-Catenin HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho ß-Catenin (Thr41/Ser37/Ser33) 2-plate assay protocol

Phospho ß-Catenin (Thr41/Ser37/Ser33) 1-plate assay protocol

Detection of Phosphorylated Phospho ß-Cateninwith 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.
Phospho ß-Catenin (Thr41/Ser37/Ser33) 1-plate protocol

HTRF assay compared to Western blot using phospho-ß-Catenin and total ß-Catenin cellular assays

Human HEK293 cells were grown in a T175 flask at 37°C, 5% CO2 until 80% confluence and treated with MG132 at 5µM (3h). Cell culture medium was discarded, and 3 mL of supplemented lysis buffer were added for a 30 minutes incubation at room temperature. 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 dispensed and analyzed side-by-side by Western Blot and by HTRF. The HTRF phospho-ß-Catenin (T41/S33/S37) assay is as sensitive as WB, whereas HTRF total ß-Catenin is 9-fold more sensitive than the Western Blot.
HTRF vesus Western blot using the phospho and total ß-Catenin assays

BIO dose-response, a GSK3 inhibitor, on HEK293 cells using phospho-beta-Catenin and total beta-Catenin cellular assays

200,000 human HEK293 cells were plated in 96 well plates and incubated for 24h at 37 °C - 5% CO2. The cells were then treated with MG-132 and increasing concentrations of BIO for 3h. For cells lysis, 50µl of lysis buffer were added 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-beta-Catenin and total beta-Catenin detection reagents were added. The HTRF signal was recorded after an overnight incubation.
phospho and total ß-Catenin assay validation on HEK293 cells

Compatibility of the HTRF phospho-ß-Catenin cellular assay with different cell lines

Cells were plated at different cell densities in 96 well plates and incubated for 24h at 37 °C - 5% CO2. 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-ß-Catenin detection reagents were added. The HTRF signal was recorded after an overnight incubation. HTRF quantification of the ß-Catenin expression level is in good agreement with the literature (sadot et al, Journal of cell sciences 2002). The ß-catenin content is almost 2.5 higher in the SW-480 cells compared to HCT-116 cells and 15 higher in the SW-480 cells compared to HEK-293 cells.
phospho ß-Catenin assay validation on HCT-116, SW-480, and HEK293 cells

Inhibition of proteasome activity by MG-132, measured by the HTRF phospho-ß-Catenin and total beta-Catenin cellular assays

200,000 human HEK293 cells were plated in 96 well plates and incubated for 24h at 37 °C - 5% CO2. The cells were then treated with increasing concentrations of MG-132 for different incubation time. For cells lysis, 50µl of lysis buffer were added 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-beta-Catenin and total beta-Catenin detection reagents were added. The HTRF signal was recorded after an overnight incubation.
Inhibition of proteasome activity by MG-132, measured by the HTRF phospho-ß-Catenin
Inhibition of proteasome activity by MG-132, measured by the HTRF total-ß-Catenin

The role of ß-Catenin in the canonical Wnt pathway

In the absence of extracellular WNT ligands, cytosolic ß-Catenin is phosphorylated on Thr41/Ser37/Ser33, thereby targeting the protein for ubiquitination, degradation and inactivation. Binding of extracellular WNT ligands Frizzled receptor and/or the LDL receptor related proteins LRP 5 and 6, transduces their signal intracellularly via activation of disheveled DVL proteins. This results in the inactivation of the destruction complex, allowing cytosolic, non-phosphorylated ß-Catenin to accumulate and thereafter to translocate to the nucleus.
The role of ß-Catenin in the canonical Wnt pathway
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Notes d'application

Cisbio lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

physiologically relevant results fo fast flowing research - Flyers

Save time and money

Switch to HTRF assays - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

Side-by-side comparison of HTRF, Western Blot, ELISA and AlphaScreen® SureFire®

Do all cell-based kinase assays perform similarly? - 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

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Notes d'application

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Notes d'application

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Notes d'application

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Notes techniques

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Notes d'application

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Notes techniques

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Notes techniques

How to run a cell based phospho HTRF assay

What to expect at the bench - Vidéos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Vidéos

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalogue

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

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Vidéos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Vidéos

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