Revvity, 64MK4PEH, HTRF Human Phospho-MKK4 (Ser257) Detection Kit, 10,000 Assay Points

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.
HTRF assays offer many advantages over other technologies:

Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
How it works
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
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 (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. Application-Cell Signaling
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 1 Lysis Buffer 2 Lysis Buffer 3 Lysis Buffer 4 Lysis Buffer 5
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Target Species-Human
Technology-TR-FRET
Therapeutic Area-NASH/Fibrosis Neuroscience Oncology & Inflammation
Unit Size-10,000 assay points