Revvity, 64MLKLTPEH, HTRF Human Total MLKL Detection Kit, 10,000 Assay Points

Overview
The Total-MLKL cellular assay monitors total MLKL, and can be used as a normalization assay with the Phospho-MLKL (Ser358) kit. This kit is compatible with the buffers from the phospho-MLKL kit, so the same lysate can be used for analyses of both the phosphorylated and the total protein populations.

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
Total-MLKL assay principle
The HTRF Total-MLKL assay quantifies the expression level of MLKL in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-MLKL assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of MLKL 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-MLKL 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 Total-MLKL HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Total-MLKL one-plate assay protocol
Detection of Total-MLKL 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-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-Inflammation Neuroscience Oncology & Inflammation
Unit Size-10,000 assay points