Human Apolipoprotein B kit
ApoB quantification in cells, supernatants and plasma
Phospho-FoxO1 (Ser256) cellular kit HTRF®
The phospho-FoxO1 kit is designed to monitor the inactive form of FoxO1 modulation, phosphorylated on Ser256.
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Ease-of-use
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Highly specific
Overview
The phospho-FoxO1 (Ser256) assay is designed for the robust quantification of FoxO1 modulation, phosphorylated on Ser256, as an indicator of its inactivation. FoxO1 is a member of the Forkhead transcription factor FOXO subfamily, and is highly expressed in insulin-responsive tissues where it regulates glucose/lipid metabolism and stress resistance. It also functions as a tumor suppressor by inhibiting cell proliferation. FoxO1 deregulation plays a critical role in the development of metabolic disorders such as diabetes and NAFLD. Its dysfunction is also linked to various types of cancer.
Benefits
- COMPATIBLE WITH MANY CELL TYPES
- SPECIFICITY
Phospho-FoxO1 (Ser251) assay principle
The Phospho-FoxO1 (Ser251) assay measures FoxO1 when phosphorylated at Ser251. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-FoxO1 (Ser251) 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 proteins phosphorylation state under a no-wash assay format.
Phospho-FoxO1 (Ser251) 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-FoxO1 (Ser251) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-FoxO1 (Ser251) 1-plate assay protocol
Detection of Phosphorylated FoxO1 (Ser251) 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.
FoxO1 Inactivation following insulin treatment
Huma HepG2 and mouse C2C12 cells were plated and cultured in a high-glucose medium prior to treatment with increasing concentrations of insulin for 1 hour. After lysis, cells were transferred twice over into a low volume white microplate finally before before the additon of the HTRF phospho-FoxO1 or total FoxO1 detection antibodies. The HTRF signal was recorded after 4 hours of incubation. In both cell lines, insulin treatment induces FoxO1 phosphorylation on Ser256 (inactivation), while its expression level remains stable.
FoxO1 activation in HepG2 cells with cytokines and oxidative stress
Human HepG2 cells were plated and cultured in high-glucose culture medium before being treated with a cocktail of TNF-a/IL-1ß/IL-6 or H2O2. Following lysis, soluble cell lysates were transferred twice over into a low volume white microplate before finally adding HTRF phospho-FoxO1 or total FoxO1 detection antibodies. Results indicate pro-inflammatory cytokines and ROS (reactive oxygen species) are factors driving metabolic deregulation by inducing FoxO1 dephosphorylation (activation) in HepG2 cell line, while its expression level remains stable.
FoxO1 Activaiton using PI3K inhibitor Wortmannin in pancreatic ß-cells
Mouse pancreatic ß-cell line Min-6 was plated and cultured in high-glucose culture medium for 3 days prior to treatment with increasing concentrations Wortmannin for 1 hour. Following lysis, cell lysates were transferred twice over into a low volume white microplate before finally adding HTRF phospho-FoxO1 or total FoxO1 detection antibodies. Results showed Wortmannin induces PI3K/AKT pathway inhibition, leading to FoxO1 dephosphorylation (activation) in Min-6 cell line. The FoxO1 expression level remains stable, demonstrating that there is no cytotoxic effect of the compound on the cells.
Inactivation of FoxO1 using IGF-1 in cancer cervical cell line HeLa
Human HeLa cells were plated and cultured in complete medium and incubated for 24. They were then incubated overnight in serum-free medium before treatment with increasing concentrations of IGF-1. Folowing lysis, cell lysates were transferred twice over into a low volume white microplate before the addition of phospho or total FoxO1 antibodies. Results show that the growth factor IGF-1 induces FoxO1 inactivation by phosphorylation on Ser256, leading to tumor cell proliferation and that it's expression level remains stable.
HTRF phospho- & total FoxO1 assays vs WB on human HEK293 cells
HEK293 cells were seeded and cultured in complete medium until 90% confluency was reached. Following lysis, soluble supernatants were collected via centrifugation. Serial dilutions of the cell lysate were performed and transferred into a low volume white microplate before the addition of HTRF phospho- or total detection antibodies. Equal amounts of lysates were used for a side by side comparison between WB and HTRF. The HTRF assay is shown to be 2-fold more sensitive than the Western Blot technique. Using the HTRF Total FoxO1 kit the HTRF assay is 8-fold more sensitive than the WB.
FoxO1 Simplified Pathway
The Insulin/IGF-1 signaling pathway activates the AKT kinase which in turn phosphorylates FoxO1 on Ser256, leading to its cytoplasmic sequestration and the inhibition of its transcriptional activity. In these conditions, hepatic glucose production is inhibited, lipogenesis increases, and cells proliferate. Conversely, the dephosphorylated form of FoxO1 can translocate to the nucleus and induce the transcription of genes involved in glucose production, lipolysis, inhibition of lipogenesis, stress resistance, apoptosis, autophagy and inflammation. The stress proteins JNK and p38 activate FoxO1 in the presence of pro-inflammatory cytokines as well as oxidative stress, fasting and exercise.
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling
Analyse of PI3K/AKT/mTor translational control pathway - Application Notes
Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening
In collaboration with Bayer - Scientific Presentations
Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers
HTRF cellular phospho-protein assays
Physiologically relevant results fo fast flowing research - Flyers
Biomarker and Cell Signaling Assays for Fibrosis and NASH
HTRF solutions for NASH - Flyers
Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers
PI3K/AKT/mTor translational control pathway - Posters
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
From 2D, 3D cell cultures to xenografts: A smart HTRF platform to maximize anticancer drug discovery
One technology across all samples - Application Notes
HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts
Tumor xenograft analysis: HTRF versus Western blot - Application Notes
FOXO1: a role in non-alcoholic fatty liver disease NASH
FOXO1 use in a fatty liver disease model - 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
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
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
A single technology for 2D cells, 3D cells, and xenograft models - Posters
Best practices for analyzing tumor xenografts with HTRF phospho assays
Protocol for tumor xenograft analysis with HTRF - Technical Notes
Webinar: NASH - Fibrosis Research and Drug Discovery
Featuring a panel of experts - Videos
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
Product Insert FoxO1 P-S256 Kit / 64FOXPEG-64FOXPEH
64FOXPEG-64FOXPEH - Product Insert
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
Advance your research on Fibrosis
Kits and reagents for Fibrosis research - Flyers
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 (ENG-US) FoxO1 P-S256 Kit / 64FOXPEG
64FOXPEG - Safety Data Sheet
Safety Data Sheet (DEU) FoxO1 P-S256 Kit / 64FOXPEG
64FOXPEG - Safety Data Sheet
Safety Data Sheet (ELL) FoxO1 P-S256 Kit / 64FOXPEG
64FOXPEG - Safety Data Sheet
Safety Data Sheet (FRA-FR) FoxO1 P-S256 Kit / 64FOXPEG
64FOXPEG - Safety Data Sheet
Safety Data Sheet (ITA) FoxO1 P-S256 Kit / 64FOXPEG
64FOXPEG - Safety Data Sheet
Safety Data Sheet (SPA) FoxO1 P-S256 Kit / 64FOXPEG
64FOXPEG - Safety Data Sheet
Safety Data Sheet (ENG-GB) FoxO1 P-S256 Kit / 64FOXPEG
64FOXPEG - Safety Data Sheet
Safety Data Sheet (DEU) FoxO1 P-S256 Kit / 64FOXPEH
64FOXPEH - Safety Data Sheet
Safety Data Sheet (ELL) FoxO1 P-S256 Kit / 64FOXPEH
64FOXPEH - Safety Data Sheet
Safety Data Sheet (FRA-FR) FoxO1 P-S256 Kit / 64FOXPEH
64FOXPEH - Safety Data Sheet
Safety Data Sheet (ITA) FoxO1 P-S256 Kit / 64FOXPEH
64FOXPEH - Safety Data Sheet
Safety Data Sheet (SPA) FoxO1 P-S256 Kit / 64FOXPEH
64FOXPEH - Safety Data Sheet
Safety Data Sheet (ENG-GB) FoxO1 P-S256 Kit / 64FOXPEH
64FOXPEH - Safety Data Sheet
Safety Data Sheet (ENG-US) FoxO1 P-S256 Kit / 64FOXPEH
64FOXPEH - Safety Data Sheet
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