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HTRF Total IR-β (Insulin Receptor Beta) Detection Kit, 500 Assay Points

This HTRF assay enables the detection of cellular Insulin Receptor beta and can be used as a normalization assay for the phospho-IR beta kit.

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Feature	Specification
Application	Cell Signaling
Sample Volume	16 µL

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This HTRF assay enables the detection of cellular Insulin Receptor beta and can be used as a normalization assay for the phospho-IR beta kit.

View product information

Product variants

Part #:

63ADK019PEG

List price

USD 2,271.53

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Part #:

63ADK019PEH

List price

USD 13,214.42

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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).

Product information

Overview

Total Insulin Receptor-ß assay enables the detection of Total Insulin Receptor-ß. Stimulated by insulin and insulin-like growth factors, the insulin receptor is involved in several pathways such as those for Insulin and FoxO signaling.

How it works

Total-Insulin Receptor ß assay principle

The Total-Insulin Receptor ß assay quantifies the expression level of Insulin Receptor ß in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-Insulin Receptor ß 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 Insulin Receptor ß 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.

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

Total-insulin receptor ß 1-plate assay protocol

Detection of total Insulin Receptor ß 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-IRβ (Tyr1150/1151) illustration on HepaRG

HepaRG cells were plated at 200,000 cells per well on a 96-well plate. After an incubation of 5 days at 37°C, 5% CO2, a serial dilution of insulin was added under cells in presence of pervanadate at 30µM for 5 minutes at 37°C, 5% CO2. Stimulation medium was removed from cells and 50µL of lysis buffer was added onto the cells. Lysis step was done by shaking gently during 30 minutes. 16µL of samples were transferred in a 384-well small volume plate then 4µL of phospho IRβ HTRF detection reagents were added. In parrallel 16µl were dispensed on other wells than 4µL of total IRβ HTRF detection reagents were added. Signals were recored overnight.

Total-IRβ WB comparison

Human Hek 293 cells were cultured to 80% confluency. After insulin and pervanadate co-treatment, cells were lysed and soluble supernatants were collected via centrifugation. Serial dilutions of the cell lysate were performed and 16 µL of each dilution were transferred into a 384-well low volume white microplate before finally adding phospho IRβ HTRF cellular kit reagents. A side by side comparison showed the HTRF Phospho assay is at least 8-fold more sensitive than the Western Blot.

Specifications

Other specifications

Application	Cell Signaling
Brand	HTRF
Detection Modality	HTRF
Molecular Modification	Total
Product Group	Kit
Sample Volume	16 µL
Shipping Conditions	Shipped in Dry Ice
Target Class	Phosphoproteins
Technology	TR-FRET
Therapeutic Area	Metabolism/Diabetes Neuroscience
Unit Size	500 assay points