Human Mixed Lineage Kinase Domain Like Protein (MLKL) ELISA Kit

Principle of the Assay

The microtiter plate provided in this kit has been pre-coated with an antibody specific to MLKL. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to MLKL. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After the TMB substrate solution is added, only those wells that contain MLKL, biotin-conjugated antibody, and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution, and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of MLKL in the samples is then determined by comparing the O.D. of the samples to the standard curve.

For Use with serum, plasma, and cell culture supernatants. For Research Use Only. Not for use in diagnostic procedures.

Target Information

Pseudokinase that plays a key role in TNF-induced necroptosis, a programmed cell death process (PubMed:22265413, PubMed:22265414, PubMed:22421439, PubMed:24316671). Does not have protein kinase activity (PubMed:22265413, PubMed:22265414, PubMed:22421439, PubMed:24316671). Activated following phosphorylation by RIPK3, leading to homotrimerization, localization to the plasma membrane and execution of programmed necrosis characterized by calcium influx and plasma membrane damage (PubMed:22265413, PubMed:22265414, PubMed:22421439, PubMed:24316671). In addition to TNF-induced necroptosis, necroptosis can also take place in the nucleus in response to orthomyxoviruses infection: following activation by ZBP1, MLKL is phosphorylated by RIPK3 in the nucleus, triggering disruption of the nuclear envelope and leakage of cellular DNA into the cytosol.following ZBP1 activation, which senses double-stranded Z-RNA structures, nuclear RIPK3 catalyzes phosphorylation and activation of MLKL, promoting disruption of the nuclear envelope and leakage of cellular DNA into the cytosol (By similarity). Binds to highly phosphorylated inositol phosphates such as inositolhexakisphosphate (InsP6) which is essential for its necroptotic function (PubMed:29883610).

GENE ID	197259
SWISS PROT	Q8NB16
SYNONYMS

Materials Supplied

Kit Components	96 Wells Quantity/Size
Pre-coated, ready-to-use 96-well strip plate	1 plate
Plate sealer for 96 wells	2
Standard	2 tubes
Diluent buffer	1 bottle
Detection Reagent A	1 bottle
Detection Reagent B	1 bottle
TMB Substrate	1 tube
Stop Solution	1 tube
Wash Buffer (30 ℅ concentrate)	1 tube
Product data sheet	1 copy

Storage

The TMB Substrate, Wash Buffer (30X concentrate), and the Stop Solution should be stored at 4°C upon receipt, while the other items should be stored at -20°C.

Performance Characteristics

REPEATABILITY	Intra-assay Precision (Precision within an assay): 3 samples with low, middle, and high-level MLKL were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, middle, and high-level MLKL were tested on 3 different plates, with 8 replicates in each plate. CV(%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%
SENSITIVITY	The minimum detectable dose was 11.6pg/mL.
ASSAY RANGE	31.2-2000pg/mL
SPECIFICITY	This assay has high sensitivity and excellent specificity for the detection of MLKL. No significant cross-reactivity or interference between MLKL and analogs was observed. Note: Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between MLKL and all analogs, therefore, cross reactivity may still exist.