Revvity, 64NK4PEG, HTRF Human Total MKK4 Detection Kit, 500 Assay Points

Overview
HTRF 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 buffers from phospho-MKK4 kit, so same lysate can be used analyses of both phosphorylated and 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 activation of JNK which regulates a range of biological processes implicated in tumorigenesis, neurodegenerative disorders, and fibrosis.
How it works
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. 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-Total
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-500 assay points