Phospho-ATF2 (Thr71) cellular kit
Phospho-MKK4 (Ser257) cellular kit HTRF®
This HTRF kit enables the cell-based quantitative detection of phosphorylated MKK4 (also named SEK1) as a readout multiple for MKKKs, such as ASK1, MEKKs, MLKs, Tpl2, and TAK1, involved in the upstream activation of the MKK4/JNK signaling pathway.
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High sensitivity
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All inclusive kit
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No-wash
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Low sample consumption
Overview
This HTRF cell-based assay conveniently and accurately quantifies phosphorylated MKK4 (Mitogen-Activated Protein Kinase Kinase 4) at Ser257. Phosphorylation of MKK4 on Serine 257 is induced by various MKKKinases such as TAK1, Tpl2, or ASK1, in response to cellular stresses and proinflammatory cytokines. Once phosphorylated, MKK4 preferentially triggers the activation of JNK, which regulates a range of biological processes implicated in tumorigenesis, neurodegenerative disorders, and fibrosis.
Benefits
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Phospho-MKK4 (Ser257) assay principle
The Phospho-MKK4 (Ser257) assay measures MKK4 when phosphorylated at Ser257. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses 2 antibodies, one labeled with a donor fluorophore and the other 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 independently 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-MKK4 (Ser257) two-plate assay protocol
Phospho-MKK4 (Ser257) one-plate assay protocol
Anisomycin dose-response on Retinoic Acid (RA) differentiated SH-SHY5Y cells
Human SH-SY5Y cells were plated at 25,000 cells/well in a 96-well plate. After 24 h incubation at 37 °C, 5% CO2, the cells were incubated in differentiation medium containing 10 µM of RA for 1 week in the dark. Note that due to the poor stability of RA, cell culture medium containing fresh RA was renewed daily. Once differentiated, SH-SY5Y were stimulated with increasing concentrations of anisomycin for 45 min. Then the medium was removed and 50 µL of supplemented lysis buffer 1X were added. After 30min lysis at RT under gentle shaking, 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho-MKK4 (Ser257) or total MKK4 detection reagents were added. The HTRF signal was recorded after an overnight incubation.
As described elsewhere, a dose dependent phosphorylation of MKK4 by Ser257 was induced by anisomycin, whereas total MKK4 slightly decreased under the same experimental conditions.
H2O2 stimulation on Retinoic Acid (RA) differentiated SH-SHY5Y cells
Human SH-SY5Y cells were plated at 400,000 cells/well in a 6-well plate. After 24 h incubation at 37 °C, 5% CO2, the cells were incubated in differentiation medium containing 10 µM of RA for 1 week in the dark. Note that due to the poor stability of RA, cell culture medium containing fresh RA was renewed daily. Once differentiated, SH-SY5Y were stimulated with 0.5 mM of H2O2 for 1h. Then the medium was removed and 500 µL of supplemented lysis buffer 1X were added. After 30 min lysis at RT under gentle shaking, 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho-MKK4 (Ser257) or total MKK4 detection reagents were added. The HTRF signal was recorded after an overnight incubation.
H2O2 induced phosphorylation MKK4 on Ser257 residue whereas the MKK4 expression level remained almost stable under the same experimental conditions.
Anisomycin dose-response on HepG2 cells correlated with Western Blot
Human HepG2 cells were plated at 100,000 cells/well in a 96-well plate. After an incubation of 24 h at 37 °C, 5% CO2, the cells were stimulated with increasing concentrations of anisomycin for 45min. Then the medium was removed and 50 µL of supplemented lysis buffer 1X were added. After 30 min lysis at RT under gentle shaking, 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho-MKK4 (Ser257) or total MKK4 detection reagents were added. The HTRF signal was recorded after an overnight incubation. The same amount of lysate was analyzed by Western Blot in a side by side experiment.
As shown on the graphs, both HTRF and Western Blot indicated an increase of MKK4 phosphorylation associated with a decrease of MKK4 expression.
HTRF phospho-MKK4 (Ser257) cellular assay compared to Western Blot
The human HepG2 cell line was seeded in a T175 flask and incubated a 37 °C, 5% CO2. The cells were then stimulated with Sorbitol (1 M) for 30 min before lysis.
Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF phospho-MKK4 (Ser257) detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.
Using the HTRF phospho-MKK4 (Ser257) assay, 310 cells/well were enough to detect a signal, while 5,000 cells were needed using Western Blot which relies on a Chemiluminescence signal. These results indicate that the HTRF phospho-MKK4 (Ser257) assay is 16 times more sensitive than the Western Blot.
Simplified pathway for MKK4 assays
MKK4 (Mitogen-activated Kinase Kinase 4) is a member of MAP kinase kinase family that is activated by phosphorylation on Ser257 following activation of different MKKKs, for example TAK1 or ASK1, in response to stimuli such as GPCR activation, Growth factors, cellular stresses, or inflammatory cytokines. In turn, activated MKK4 phosphorylates JNK or p38 in order to activate c-jun, p53, or ATF2 and induce inflammation, cell survival/apoptosis, proliferation, or differentiation by regulating gene transcriptions.
HTRF Product Catalog 2020 July update
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
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
Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers
Cisbio lysis buffer compatibility
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
Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences
Insider Tips for successful sample treatment - Technical Notes
Optimize your HTRF cell signaling assays on tissues
HTRF and WB compatible guidelines - Technical Notes
Best practices for analyzing tumor xenografts with HTRF phospho assays
Protocol for tumor xenograft analysis with HTRF - Technical Notes
Key guidelines to successful cell signaling experiments
Mastering the art of cell signaling assays optimization - Guides
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
HTRF® cell signaling platform combined with iCell® Hepatocytes
A solution for phospho-protein analysis in metabolic disorders - Posters
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
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
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
HTRF Alpha-tubulin Housekeeping kit
Properly interpret your compound effect - Application Notes
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
How to run a cell based phospho HTRF assay
What to expect at the bench - Videos
Product Insert MKK4 P-S257 Kit / 64MK4PEG-64MK4PEH
64MK4PEG-64MK4PEH - Product Insert
Molecular basis of neuroinflammation and neurodegeneration diseases
The essential guide for extending your knowledge on the molecular mechanisms of neurodegenerative diseases - Guides
Neurodegeneration and its main related diseases
Discover this infographic design on neurodegenerative diseases - Infographics
Assays for neurosciences research
Advance your research on neurodegenerative diseases - Flyers
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