HTRF Human & Mouse Total β-Catenin Detection Kit, 500 Assay Points

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HTRF Human & Mouse Total β-Catenin Detection Kit, 500 Assay Points

The total beta-Catenin kit monitors cytosolic total beta-Catenin and can be used as a normalization assay for the phospho beta-Catenin kit.

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption and disposal requirements under European REACH regulations (EC 1907/2006).

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Feature	Specification
Application	Cell Signaling
Sample Volume	16 µL

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The total beta-Catenin kit monitors cytosolic total beta-Catenin and can be used as a normalization assay for the phospho beta-Catenin kit.

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Product Variants

Unit Size: 500 Assay Points

Part #:

64NCAPEG

List Price

USD 2,147.00

Unit Size: 10,000 Assay Points

Part #:

64NCAPEH

List Price

USD 12,490.00

Product information

Overview

The Total ß-Catenin cellular assay offers a robust monitoring of total ß-Catenin, and is used as a normalization assay with the phospho ß-Catenin kit. Total ß-Catenin is an important co-transcriptional activator of many significant genes involved in embryonic development, adult homeostasis, and cell proliferation.

Specifications

Other Specifications

Application	Cell Signaling
Brand	HTRF
Detection Modality	HTRF
Lysis Buffer Compatibility	Lysis Buffer 3 Lysis Buffer 4 Lysis Buffer 5
Molecular Modification	Total
Product Group	Kit
Sample Volume	16 µL
Shipping Conditions	Shipped in Dry Ice
Target Class	Phosphoproteins
Target Species	Human Mouse
Technology	TR-FRET
Therapeutic Area	Metabolism/Diabetes Oncology & Inflammation
Unit Size	500 Assay Points

Video gallery

How it works

Total-ß-Catenin assay principle

The Total-ß-Catenin assay quantifies the expression level of ß-Catenin in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-ß-Catenin assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of ß-Catenin in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the proteins expression under a no-wash assay format.

phospho-how-it-works-total-beta-catenin-assay-principle

Total-ß-Catenin 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 Total ß-Catenin HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

total-beta-catenin-thr41-ser37-ser33-1-plate-protocol

Total-ß-Catenin 1-plate assay protocol

Detection of total ßCatenin 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.

total-beta-catenin-thr41-ser37-ser33-2-plate-assay-protocol

Assay validation

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.

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.

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.

Simplified pathway

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.