Revvity, 64MLKLS8PEH, HTRF Human Phospho-MLKL (Ser358) Detection Kit, 10,000 Assay Points

Overview
This HTRF cell-based assay conveniently and accurately detects phosphorylated MLKL at Ser358.
MLKL (Mixed lineage kinase domain-like protein) is a key mediator of apoptotic and necrotic cell death, as well as of inflammatory pathways downstream from TNFR1 and other receptors. MLKL is phosphorylated at Serine 358 by RIPK3, and is considered to be a biomarker of the initiation of RIPK1/RIPK3-dependent necroptosis.
Necroptosis is a programmed necrotic cell death pathway, also called "inflammatory cell death", which is closely associated with pathologies including inflammatory and neurodegenerative diseases, as well as cancer. MLKL has therefore become an important drug target for the pharmaceutical industry. Therapeutic strategies consist in developing specific small-molecule kinase inhibitors, such as the well-known Necrostatin-1 and Necrostatin-1s.
How it works
Phospho-MLKL (Ser358) assay principle
The Phospho-MLKL (Ser358) assay measures MLKL when phosphorylated at Ser358. 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-MLKL (Ser358) two-plate assay protocol
The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of Phospho-MLKL (Ser358) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Phospho-MLKL (Ser358) one-plate assay protocol
Detection of Phosphorylated MLKL (Ser358) 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
Automation Compatible-Yes
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 1 Lysis Buffer 2 Lysis Buffer 4
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-Inflammation Neuroscience Oncology & Inflammation
Unit Size-10,000 assay points