HTRF Human Total MKK4 Detection Kit, 500 Assay Points

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 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 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.

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.

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.

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.

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.