Phospho-β-Catenin (Thr41/Ser37/Ser33) & Total β-Catenin Cellular Assay Kits

Phospho- and total β-Catenin detection as a readout of the canonical Wnt pathway

Cisbio's cell-based homogeneous HTRF® assays, phospho-β-Catenin (Thr41/Ser37/Ser33) and total β-Catenin, enable a canonical Wnt pathway readout. The phospho-β-Catenin assay provides the quantitative detection of GSK3 β-induced phosphorylation of β-Catenin on Thr41/Ser37/Ser33, which represents the inactive form of the protein degraded by the proteasome. Cytosolic total β-Catenin is an important co-transcriptional activator of many significant genes involved in embryonic development, adult homeostasis and cell proliferation. The total β-Catenin assay serves as a normalization control of the phosphorylated form and facilitates analysis of the steady state level between the 2 pools of β-Catenin – phosphorylated inactive and the non-phosphorylated forms – with their different cellular functions. Membrane-associated β –Catenin involved in cadherin-based adherens junctions cannot be discriminated by the total assay. The phospho- β-Catenin assay is thus suitable for cell-based screening of GSK3β inhibitors or other modulators of the Wnt pathway. The simple add-and-read protocol eliminates all wash steps for a faster analysis and easy miniaturization while maintaining a high-quality specific output, and offers enhanced convenience over ELISA or WB.

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Assay Principle

HTRF® - the homogeneous cell-based sandwich immunoassay

These phospho and total beta-Catenin assays are based on a TR-FRET sandwich immunoassay format comprising two specific monoclonal anti-beta-Catenin antibodies, one labeled with Eu3+-cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind β-Catenin, and the proximity of donor and acceptor will then lead to a fluorescent TR-FRET signal. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

The assays can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho- or total-beta-Catenin can be quantitatively detected using the HTRF phospho-β-Catenin (T41/S33/S37) and total β-Catenin cellular assay kit reagents and most TR-FRET multimode plate readers.

A simpler, more flexible assay protocol - adapted to your applications

Two-plate assay protocol

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated and treated in a 96-well culture plate. For detection, lysates are subsequently transferred to a 384sv assay plate where the HTRF® reagents are added. This also enables the cells' viability and confluence to be monitored in an appropriate cell culture plate.

One-plate assay protocol

This protocol can be further streamlined to a one-plate assay in which plating, treating and detecting are all performed in a single plate. No wash steps are required. Ideal for HTS - this protocol provides enhanced speed and simplicity, enabling all throughputs and fast results while maintaining a high-quality sensitive output.

What to expect at the bench

This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

Simplified pathway

The role of β-Catenin in the canonical Wnt pathway

In the absence of extracellular WNT ligands, cytosolic β-Catenin is recognized by the destruction complex comprising Axin, APC, casein kinase-1 CK-1 and glycogen synthase kinase-3 GSK-3. This destruction complex and in particular GSK3β phosphorylates β-Catenin on Thr41/Ser37/Ser33, thereby targeting the protein for ubiquitination and subsequent proteosomal degradation. Phosphorylated β-Catenin is hence the inactive form of the protein in the Wnt pathway. Upon binding of extracellular WNT ligands to the seven-transmembrane receptor Frizzled and/or the low density lipoprotein receptor related proteins LRP 5 and 6, the activated receptors transduce their signal intracellularly via activation of dishevelled DVL proteins. This results in the inactivation of the destruction complex, allowing cytosolic, non-phosphorylated β-Catenin to accumulate and thereafter to translocate to the nucleus. The transcriptional co-activator β-Catenin associates with the T-cell factor TCF/Lymphoid enhancer factor LEF family of transcription factors, and induces gene transcription of significant genes involved in embryonic development, adult homeostasis and cell proliferation (e.g. cMyc, cyclin D1). Aberrant activation of the canonical WNT signaling can result in the development of cancers, like colorectal cancers, hepatocellular carcinomas, a wide variety of solid tumors, and leukemia. The inactivation of canonical WNT signaling may lead to osteoporosis, bone-related disorders or chronic obstructive pulmonary diseases. Embryonic inactivation is lethal. The canonical WNT pathway thus represents a potentially attractive target for the treatment of these diseases.

Assay Performance

1. HTRF assay compared to Western blot using phospho-β-Catenin and total β-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 3ml of supplemented lysis buffer were added for a 30 minute 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-β-Catenin (T41/S33/S37) assay is as sensitive as WB, whereas HTRF® total β-Catenin is 9-fold more sensitive than the Western Blot.

2. 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.

3. Compatibility of the HTRF phospho-β-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-β-Catenin detection reagents were added. The HTRF signal was recorded after an overnight incubation.

HTRF quantification of the β-Catenin expression level is in good agreement with the literature (sadot et al, Journal of cell sciences 2002). The β-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.

4. Inhibition of proteasome activity by MG-132, measured by the HTRF phospho-β-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.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
Beta Catenin phospho-T41/ S33/37 kit- 500 tests64CATPEG
Beta Catenin phospho-T41/ S33/37 kit- 10,000 tests64CATPEH
Beta Catenin phospho-T41/ S33/37 kit- 50,000 tests64CATPEY
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Beta Catenin total kit - 500 tests64NCAPEG
Beta Catenin total kit - 10,000 tests64NCAPEH

Companion products

DescriptionCat. noProduct insertMSDS
Beta Catenin phospho-T41/ S33/37 control lysate64CATTDA
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Phospho-total protein blocking reagent - 2 ml64KB1AAC
Phospho-total protein blocking reagent - 6 ml64KB1AAD
Phospho-total protein lysis buffer #3 - 130 mL64KL3FDF
Beta Catenin total kit control lysate64NCATDA
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