PAb Antiphosphothreonine-Eu cryptate
Antiphosthreonine;Eu cryptate to detect Threonine kinases.
The HTRF phospho-IRS1 (Ser307) assay enables the cell-based detection of Ser307 phosphorylation on IRS1.
This HTRF cell-based assay conveniently and accurately quantifies phosphorylated IRS1 at (Ser307 mouse/Ser312 human). IRS1 is a relevant marker for insulin resistance, tumor development, and metastatic progression. It is associated with type 2 diabetes, obesity, and cancer.
The phospho-IRS1(Ser307 mouse/Ser312 human) assay measures IRS when phosphorylated at Ser307 mouse/Ser312 human. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The phospho-IRS1(Ser307 mouse/Ser312 human) 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.
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-IRS1 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Detection of phosphorylated IRS1 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.
C2C12 cells were cultured until 100% confluency was reached. They were then cultured for 7 days in 2% horse serum media to induce the differentiation. After a starvation step in serum-free medium, cells were treated with a cocktail of pro-inflammatory cytokines and/or Insulin. After cell supernatant removal, cells were lysed with 1.5 mL of supplemented lysis buffer #1 for 30 min at RT under gentle agitation. 16 µL of lysate were then transferred into a 384-well low volume white microplate for HTRF detection using the phospho- and total IRS1 kit reagents.
3T3-L1 cells were cultured in medium with 10% newborn calf serum. They were then cultured for 7 days in medium containing 10% FCS, insulin, dexamethasone, IBMX and µM thiazolidinedione to induce differentiation from fibroblasts to adipocytes. After an overnight starvation step in serum-free medium, the cells were treated with 100 nM insulin for 45 min or with a cocktail of pro-inflammatory cytokines for 30 min. After cell supernatant removal, cells were lysed and transferred into a 384-well low volume white microplate for HTRF detection using the phospho- and total IRS1 kit reagents (Samples were kindly provided by JF Tanti’s research team, UMR INSERM U1065/UNS, C3M, Nice, France).
Insider Tips for successful sample treatment - Technical Notes
HTRF and WB compatible guidelines - Technical Notes
Protocol for tumor xenograft analysis with HTRF - Technical Notes
Mastering the art of cell signaling assays optimization - Guides
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
A solution for phospho-protein analysis in metabolic disorders - Posters
Detailed protocol and direct comparison with WB - Posters
A single technology for 2D cells, 3D cells, and xenograft models - Posters
PI3K/AKT/mTor translational control pathway - Posters
Analysis of a large panel of diverse biological samples and cellular models - Posters
One technology across all samples - Application Notes
Tumor xenograft analysis: HTRF versus Western blot - Application Notes
Valuable guidelines for efficiently analyzing and interpreting results - Application Notes
Increased flexibility of phospho-assays - Application Notes
Analyse of PI3K/AKT/mTor translational control pathway - Application Notes
Do all cell-based kinase assays perform similarly? - Posters
In collaboration with Bayer - Scientific Presentations
A fun video introducing you to phosphorylation assays with HTRF - Videos
Get the brochure about technology comparison. - Brochures
Physiologically relevant results fo fast flowing research - Flyers
All-in-one HTRF kit for robust detection of Phospho-IRS1
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