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Total IR beta cellular kit HTRF®

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

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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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.


This kit has been pre-validated for use in combination with Pelago Bioscience’s CETSA® (Cellular Thermal Shift Assay) technology. Please note that further assay optimization will be required in order to adapt the kit to your particular cell line and experimental conditions. A valid CETSA® licence from Pelago Bioscience or a single kit agreement is required for using the CETSA® technology.

Benefits

  • SPECTIFICITY
  • PRECISION
  • DATA NORMALIZATION

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 protein’s expression under a no-wash assay format.
Total Insulin Receptor ß cellular assay principle

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 2-plate assay protocol

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 Insulin Receptor ß 1-plate protocol

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.
: Insulin stimulation on HepaRG cells leads phosphorylation on insulin receptor on tyrosine 1150/1151 residue

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.
HTRF assay compared to Western Blot using total-IRb cellular assay on human Hek293 cells

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

HTRF Product Catalog

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

Product Insert IRb total Kit / 63ADK019PEG-63ADK019PEH

63ADK019PEG-63ADK019PEH - Product Insert

Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection

Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes

Assessment of drug efficacy and toxicity by combining innovative technologies

Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Application Notes

Safety Data Sheet IRb total Kit / 63ADK019PEH

63ADK019PEH - Safety Data Sheet

Plate Reader Requirement

Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.

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