This HTRF kit is designed to monitor the expression level of cellular MKK4, and can be used as a normalization assay for the phospho-MKK4 kit.

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

This HTRF kit is designed to monitor the expression level of cellular MKK4, and can be used as a normalization assay for the phospho-MKK4 kit.

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Overview

Cisbio's Total MKK4 cellular assay monitors total MKK4, and can be used as a normalization assay with our phospho-MKK4 kit. This kit is compatible with the buffers from the phospho-MKK4 kit, so the same lysate can be used for analyses of both the phosphorylated and the total protein populations.

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 prefentially triggers the activation of JNK which regulates a range of biological processes implicated in tumorigenesis, neurodegenerative disorders, and fibrosis.

Benefits

  • SPECIFICITY
  • PRECISION
  • DATA NORMALIZATION

Total-MKK4 assay principle

The Total MKK4 assay quantifies the expression level of MKK4 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-MKK4 assay uses two labeled antibodies, one coupled to a donor fluorophore and the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of MKK4 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 MKK4 assay principle

Total-MKK4 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 Total-STING HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total MKK4 2-plate Assay protocol

Total-MKK4 one-plate assay protocol

Detection of Total MKK4 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 MKK4 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 10µM 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 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.

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 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.

H2O2 induced phosphorylation MKK4 on Ser257 residue, whereas the MKK4 expression level remained almost stable under the same experimental conditions.

400,000 RA differentiated 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 45 min. 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 in 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 total MKK4 cellular assay compared to Western Blot

The human HepG2 cell line was seeded in a T175 flask and incubated at 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 in the 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 total MKK4 detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

Using the HTRF total MKK4 assay, 1,250 cells/well were enough to detect a signal while 10,000 cells were needed using Western Blot which relies on an ECL detection. These results demonstrate that the HTRF total MKK4 assay is 8 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 total Kit / 64NK4PEG-64NK4PEH

64NK4PEG-64NK4PEH - Product Insert

Molecular basis of neuroinflammation and neurodegeneration diseases

The essential guide for extending your knowledge on the molecular mechanisms of neurodegenerative diseases - Guides

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