HTRF Phospho-IR-β (Insulin Receptor Beta) (Tyr1150 /1151) Detection Kit

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.

Specifications

How it works

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-beta-ser177-181-assay-principle

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-irbeta-ser177-181-2-plate-assay-protocol

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.

phospho-irbeta-ser177-181-1-plate-protocol

Assay validation

Phospho-IRβ (Tyr1150/1151) illustration on HepG2 cells

HepG2 cells were plated at 100,000 cells per well on a 96-well plate. After an overnight incubation at 37°C, 5% CO2, a serial dilution of insulin was added under cells 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. Signals were recorded overnight.

Phospho-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 parallel 16µl were dispensed on other wells than 4µL of total IRβ HTRF detection reagents were added. Signals were recorded overnight.

assay-validation-irb-phospho-y1150-1151-2

Phospho-IRβ (Tyr1150/1151) 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.

assay-validation-irb-phospho-y1150-1151-3

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Cat.No	Name	Size
63ADK016PEH	HTRF IR BETA P-Y1150KIT	10K PTS