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 High sensitivity
  • All inclusive kit All inclusive kit
  • No-wash No-wash
  • Low sample consumption Low sample consumption

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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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

  • SPECIFICITY
  • PRECISION

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-S257 Assay principle

Phospho-MKK4 (Ser257) two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a 384-well low volume detection plate before the addition of Phospho-MKK4 (Ser257) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-MKK4 S257 2-plate Assay protocol

Phospho-MKK4 (Ser257) one-plate assay protocol

Detection of Phosphorylated MKK4 (Ser257) 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-MKK4 S257 1-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.

Anisomycin dose-response on 25,000 RA differentiated SH-SY5Y cells/ well

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.

400,000 RA differentitated SH-SY5Y cells were treated with 0.5mM of H202

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 results after anisomycin dose-response on 100,000 HepG2 cells/ well
Western Blot results after anisomycin dose-response on 100,000 HepG2 cells/ well

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.

Western Blot and HTRF sensitivity comparison on HepG2 control lysate

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.

MAPK/JNK and MAPK/p38 signaling pathways
Cisbio Product Catalog 2019

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

Species compatibility

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

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

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

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

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

Side-by-side comparison of HTRF, Western Blot, ELISA and AlphaScreen® SureFire®

Do all cell-based kinase assays perform similarly? - Posters

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

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