HMGB1 kit
HTRF Total MLKL Cellular Kit HTRF®
The Total MLKL kit is designed to monitor the expression level of cellular MLKL, and can be used as a normalization assay for the Phospho-MLKL (Ser358) kit.
-
No-wash
-
High sensitivity
-
All inclusive kit
-
Low sample consumption
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.
Benefits
- SPECIFICITY
- PRECISION
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.
MLKL activation in TNFa stimulated HT-29 cells
The human colorectal cancer cell line HT-29 was seeded in a 96-well culture-treated plate under 200,000 cells/well in complete culture medium, and incubated overnight at 37 °C, 5% CO2. For MLKL activation experiments, the cells were pre-treated with 25 µM Z-VAD (pan-caspase inhibitor) for 30 minutes before the simultaneous addition of SM-164 (cIAP1/2 inhibitor) at 100 nM and human TNF-α at various concentrations for 6 hours. After treatment, the cells were lyzed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking. For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Phospho-MLKL (Ser358) or Total-MLKL detection reagents were added. The HTRF signal was recorded after an overnight incubation.
In presence of SM-164 and Z-VAD, that block cell survival and apoptosis respectively, TNF-α induces a dose-dependent increase in MLKL phosphorylation at Ser358, highlighting the activation of the necroptosis pathway. Total MLKL show unchanged levels of signal, demonstrating that the modulations observed on Phospho-MLKL is induced by the pharmacological treatments, and is not caused by cell detachment that can happen during late stages of necroptosis.
Prevention of necroptosis activation in HT-29 cells using necroptosis inhibitors
The human colorectal cancer cell line HT-29 was seeded in a 96-well culture-treated plate under 200,000 cells/well in complete culture medium, and incubated overnight at 37 °C, 5% CO2. The cells were pre-treated with 25 µM Z-VAD for 20 minutes before the addition of increasing doses of Necrostatin-1s or Necrostatin-1. After 10 additional minutes, a pre-mix containing 100 ng/mL TNF-α and 100 nM SM-164 was added for 6h. After treatment, the cells were lyzed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking. For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate, and 4 µL of the HTRF Phospho-MLK (Ser358) or Total-MLKL detection reagents were added. The HTRF signal was recorded after an overnight incubation.
The necroptosis inhibitor Necrostatin-1 and its analog Necrostatin-1s both induce a dose-dependent decrease in MLKL phosphorylation at Ser358, while total MLKL remain stable. As expected, Necrostatin-1s is more potent than the original small molecule, based on the IC50 values obtained on Phospho-MLKL that are ~ 2 times better than with Necrostatin-1.
HTRF Total MLKL assay compared to Western Blot
The human colorectal cancer cell line HT-29 was cultured in a T175 flask in complete culture medium for 48h at 37°C, 5% CO2. The cells were pre-treated with 25 µM Z-VAD for 30 minutes before the simultaneous addition of SM-164 at 100 nM and human TNF-α at 100 ng/mL for 6 hours. After treatment, the cells were lyzed with 3 mL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking.
Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF Total MLKL detection reagents. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.
Using the HTRF Total MLKL assay, 4,100 cells/well were enough to detect a significant signal, while 8,125 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore, in these conditions, the HTRF Total MLKL assay was 2 times more sensitive than the Western Blot technique.
MLKL Signaling Pathway
MLKL plays key role in TNF-a induced necroptosis, programmed cell death process.
Upon binding of TNF-α to TNFR1, the receptor triggers the rapid formation of complex I at the cytoplasmic membrane by recruiting multiple proteins, including RIPK1, TRADD, TRAF2, and the cellular inhibitors of apoptosis 1 and 2 (cIAP1/2). cIAP1/2 catalyze the polyubiquitination of RIPK1, leading to the recruitment of IKKα and IKKβ and the activation of the NF-κB pro-survival pathway.
In the absence of cIAP1/2, RIPK1 is released from TNFR1 and associates with FADD and caspase 8 to form the cytosolic complex IIa, responsible for caspase 8-dependent cell apoptosis.
When caspase 8 activity is inhibited or under pathological conditions, MLKL interacts with RIPK1 and RIPK3 to form the cytosolic complex IIb (also called 'necrosome') which is involved in the initiation of necroptosis. Following RIPK1 autophosphorylation MLKL is phosphorylated by RIPK3 at Ser358 leading to its oligomerization, localization to the plasma membrane and execution of programmed necrosis characterized by calcium influx and plasma membrane damage . DAMPs are then released to promote damage repairs.
HTRF cellular phospho-protein assays
Physiologically relevant results fo fast flowing research - Flyers
Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences
Insider Tips for successful sample treatment - Technical Notes
Optimize your HTRF cell signaling assays on tissues
HTRF and WB compatible guidelines - Technical Notes
Best practices for analyzing tumor xenografts with HTRF phospho assays
Protocol for tumor xenograft analysis with HTRF - Technical Notes
Key guidelines to successful cell signaling experiments
Mastering the art of cell signaling assays optimization - Guides
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
HTRF® cell signaling platform combined with iCell® Hepatocytes
A solution for phospho-protein analysis in metabolic disorders - Posters
HTRF phospho-assays reveal subtle drug-induced effects
Detailed protocol and direct comparison with WB - Posters
A single technology for 2D cells, 3D cells, and xenograft models - Posters
PI3K/AKT/mTor translational control pathway - Posters
Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells
Analysis of a large panel of diverse biological samples and cellular models - Posters
From 2D, 3D cell cultures to xenografts: A smart HTRF platform to maximize anticancer drug discovery
One technology across all samples - Application Notes
HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts
Tumor xenograft analysis: HTRF versus Western blot - Application Notes
HTRF cell-based phospho-protein data normalization
Valuable guidelines for efficiently analyzing and interpreting results - Application Notes
HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers
Increased flexibility of phospho-assays - Application Notes
HTRF Alpha-tubulin Housekeeping kit
Properly interpret your compound effect - Application Notes
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling
Analyse of PI3K/AKT/mTor translational control pathway - Application Notes
How to run a cell based phospho HTRF assay
What to expect at the bench - Videos
Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening
In collaboration with Bayer - Scientific Presentations
Unleash the potential of your phosphorylation research with HTRF
A fun video introducing you to phosphorylation assays with HTRF - Videos
How to run a cell based phospho HTRF assay
3' video to set up your Phospho assay - Videos
Product Insert MLKL total Kit / 64MLKLTPEG-64MLKLTPEH
64MLKLTPEG-64MLKLTPEH - Product Insert
Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection
Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes
Assessment of drug efficacy and toxicity by combining innovative technologies
Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes
Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)
Learn how to reduce time and sample consumption - Application Notes
Safety Data Sheet (DEU) MLKL total Kit / 64MLKLTPEG
64MLKLTPEG - Safety Data Sheet
Safety Data Sheet (ELL) MLKL total Kit / 64MLKLTPEG
64MLKLTPEG - Safety Data Sheet
Safety Data Sheet (FRA-FR) MLKL total Kit / 64MLKLTPEG
64MLKLTPEG - Safety Data Sheet
Safety Data Sheet (ITA) MLKL total Kit / 64MLKLTPEG
64MLKLTPEG - Safety Data Sheet
Safety Data Sheet (SPA) MLKL total Kit / 64MLKLTPEG
64MLKLTPEG - Safety Data Sheet
Safety Data Sheet (ENG-GB) MLKL total Kit / 64MLKLTPEG
64MLKLTPEG - Safety Data Sheet
Safety Data Sheet (ENG-US) MLKL total Kit / 64MLKLTPEG
64MLKLTPEG - Safety Data Sheet
Safety Data Sheet (DEU) MLKL total Kit / 64MLKLTPEH
64MLKLTPEH - Safety Data Sheet
Safety Data Sheet (ELL) MLKL total Kit / 64MLKLTPEH
64MLKLTPEH - Safety Data Sheet
Safety Data Sheet (FRA-FR) MLKL total Kit / 64MLKLTPEH
64MLKLTPEH - Safety Data Sheet
Safety Data Sheet (ITA) MLKL total Kit / 64MLKLTPEH
64MLKLTPEH - Safety Data Sheet
Safety Data Sheet (SPA) MLKL total Kit / 64MLKLTPEH
64MLKLTPEH - Safety Data Sheet
Safety Data Sheet (ENG-GB) MLKL total Kit / 64MLKLTPEH
64MLKLTPEH - Safety Data Sheet
Safety Data Sheet (ENG-US) MLKL total Kit / 64MLKLTPEH
64MLKLTPEH - Safety Data Sheet
Plate Reader Requirement
Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.
Let's find your readerYou might be interested in