This HTRF assay enables the cell-based detection of Tyr1150/1151 phosphorylation on Insulin Receptor-beta.

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  • No-wash
  • Low sample consumption
  • Faster and more convenient than ELISA

This HTRF assay enables the cell-based detection of Tyr1150/1151 phosphorylation on Insulin Receptor-beta.

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Overview

Phospho-Insulin Receptor-beta (Tyr1150/1151) assay enables the detection of Tyr1150/1151 phosphorylation on Insulin Receptor-beta. Stimulated by insulin and insulin-like growth factors, the insulin receptor is involved in several pathways such as those for Insulin and FoxO signaling.

Phospho-Insulin Receptor ß (Ser177/181) assay principle

The Phospho-Insulin Receptor-beta (Ser177/181) assay measures Insulin Receptor-beta when phosphorylated at Ser177/181. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-Insulin Receptor-beta (Ser177/181) 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-Insulin Receptor ß (Ser177/181) 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-IR-beta (Ser177/181) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-Insulin Receptor ß (Ser177/181) 1-plate assay protocol

Detection of Phosphorylated IR-beta (Ser177/181) 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.
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