Phospho MLKL antibody pS358 [EPR9514] ab187091 is a rabbit monoclonal antibody that is used in MLKL western blotting and IHC. Suitable for human samples.
- Recombinant format for unrivaled batch-batch consistency: no need for same-lot requests
- Specificity and sensitivity confirmed in IHC with multi-tissue microarray (TMA) validation
- Antibody clone EPR9514 is cited in over 250 publications
IgG
Rabbit
pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Liquid
Monoclonal
IHC-P | Dot | WB | |
---|---|---|---|
Human | Tested | Expected | Tested |
Mouse | Not recommended | Not recommended | Not recommended |
Rat | Not recommended | Not recommended | Not recommended |
Synthetic peptide - Human | Not recommended | Tested | Not recommended |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 1/250 - 1/500 | Notes Not Suitable for Mouse and Rat Perform heat-mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. |
Species | Dilution info | Notes |
---|---|---|
Species Mouse | Dilution info - | Notes Not Suitable for Mouse and Rat Perform heat-mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. |
Species Rat | Dilution info - | Notes Not Suitable for Mouse and Rat Perform heat-mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. |
Species Synthetic peptide - Human | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Synthetic peptide - Human | Dilution info 1/1000 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info Use at an assay dependent concentration. | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Mouse, Rat | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 1/1000 - 1/2000 | Notes We recommend using 1% SDS Hot lysis method to prepare cell lysates. For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here. |
Species | Dilution info | Notes |
---|---|---|
Species Mouse | Dilution info - | Notes We recommend using 1% SDS Hot lysis method to prepare cell lysates. For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here. |
Species Rat | Dilution info - | Notes We recommend using 1% SDS Hot lysis method to prepare cell lysates. For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here. |
Species Synthetic peptide - Human | Dilution info - | Notes - |
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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).
Mixed lineage kinase domain-like protein, hMLKL, MLKL
Phospho MLKL antibody pS358 [EPR9514] ab187091 is a rabbit monoclonal antibody that is used in MLKL western blotting and IHC. Suitable for human samples.
- Recombinant format for unrivaled batch-batch consistency: no need for same-lot requests
- Specificity and sensitivity confirmed in IHC with multi-tissue microarray (TMA) validation
- Antibody clone EPR9514 is cited in over 250 publications
IgG
Rabbit
pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Liquid
Monoclonal
EPR9514
Affinity purification Protein A
Stimulation may be required to allow detection of the phosphorylated protein. Please see images below for recommended treatment conditions and positive controls.
FURTHER INFORMATION ON SPECIFICITY (Chinese Version) available under the support & downloads section.
Blue Ice
1-2 weeks
+4°C
-20°C
Upon delivery aliquot
Avoid freeze / thaw cycle
This antibody was developed through collaboration with the lab of Xiaodong Wang at the National Institute of Biological Sciences, Beijing.
Abcam recommended secondaries - Goat Anti-Rabbit HRP (Goat Anti-Rabbit IgG H&L (HRP) ab205718) and Goat Anti-Rabbit Alexa Fluor® 488 (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077). Or search our wide range of secondary antibodies for use with your experiment.
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
This product is a recombinant monoclonal antibody, which offers several advantages including:
For more information, read more on recombinant antibodies.
This supplementary information is collated from multiple sources and compiled automatically.
MLKL also known as mixed lineage kinase domain-like protein plays a critical role in the process of necroptosis a form of programmed cell death. The MLKL protein has a molecular weight of approximately 54 kDa. The protein exists mainly within the cytoplasm but translocates to the plasma membrane during cell death execution. Expression of MLKL happens in various tissues indicating its wide biological importance. Phosphorylation of MLKL often referred to as p-MLKL is key to triggering its activity marking the transition from an inactive to an active state during necroptosis.
The MLKL protein acts as an executioner of cell death by forming a complex that disrupts the plasma membrane integrity. This process is downstream of receptor-interacting serine/threonine-protein kinase 3 (RIPK3) which phosphorylates MLKL to form the active necrosome complex. Active MLKL oligomerizes and migrates towards the inner leaflet of the plasma membrane binding to phosphatidylinositol phosphates which assists in pore formation and cellular rupture. The ability to measure MLKL activity levels such as via MLKL ELISA kits is important for understanding necrotic processes in detailed studies.
MLKL is integrally involved in the necroptotic pathway alongside RIPK1 and RIPK3 which are key initiators of necroptosis. Phosphorylated MLKL acts downstream of RIPK3 resulting in cell death without caspase activation distinguishing necroptosis from apoptosis. MLKL and RIPK3 are tightly linked within this pathway with MLKL phosphorylation serving as a vital event for the execution phase. The necroptosis pathway is part of larger networks including inflammatory response pathways highlighting the importance of MLKL's role beyond sheer cell death.
MLKL has been implicated in various inflammatory conditions and neurodegenerative diseases. The dysregulation of necroptosis can contribute to disorders such as inflammatory bowel disease and amyotrophic lateral sclerosis. In inflammatory bowel disease increased levels of p-MLKL might lead to excessive cell death exacerbating inflammation. Similarly in neurodegenerative disorders the harmful activation of MLKL may accelerate neuronal cell death. Key interactions with proteins like RIPK3 and RIPK1 highlight MLKL's involvement in these pathological processes making it a potential target for therapeutic intervention.
We have tested this species and application combination and it works. It is covered by our product promise.
We have not tested this specific species and application combination in-house, but expect it will work. It is covered by our product promise.
This species and application combination has not been tested, but we predict it will work based on strong homology. However, this combination is not covered by our product promise.
We do not recommend this combination. It is not covered by our product promise.
We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.
In the unlikely event of one of our products not working as expected, you are covered by our product promise.
Full details and terms and conditions can be found here:
Terms & Conditions.
Blocking buffer and concentration: 5% NFDM/TBST, Diluting buffer and concentration: 5% NFDM /TBST, Exposure time: 1 minute
The lysate in this image is prepared by 1%SDS Hot Lysate buffer.
For Lysate preparation protocol, please refer to the protocol section of the website and/or here.
All lanes: Western blot - Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091) at 1/1000 dilution
Lane 1: Untreated HT-29 (human colorectal adenocarcinoma) whole cell lysates 20μg
Lane 2: HT-29 (human colorectal adenocarcinoma) treated with TNF alpha+ Smac mimetic+ z-VAD whole cell lysates 20μg
Lane 3: HT-29 (human colorectal adenocarcinoma) treated with TNF alpha+ Smac mimetic + z-VAD and phosphatase whole cell lysates 20μg.
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Predicted band size: 54 kDa
Observed band size: 54 kDa
ab187091 at 1:250 staining MLKL (phospho S358) in Human melanoma tissue by immunohistochemistry (FFPE).
Antigen retrival required on FFPE tissue: HIER using 10mM Citrate buffer pH 6.0, see recommended HIER protocol
For additional IHC guideline, please see IHC resource page
ab187091 at 1:250 staining MLKL (phospho S358) in Human skin tissue by immunohistochemistry (FFPE).
Antigen retrival required on FFPE tissue: HIER using 10mM Citrate buffer pH 6.0, see recommended HIER protocol
For additional IHC guideline, please see IHC resource page
Dot blot analysis of MLKL peptides using ab187091 at 1/1000 dilution followed by Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated secondary antibody at 1/1000 dilution. Blocking and diluting buffer was 5% NFDM/TBST, exposure time 3 minuts.
Lane 1: MLKL (pT357) phospho peptide
Lane 2: MLKL (pS358) phospho peptide
Lane 3: MLKL (pT357/pS358) phospho peptide
Lane 4: MLKL non-phospho peptide
All lanes: Western blot - Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091) at 1000 Cells
Lane 1: Untreated human hepatocyte cell lysate at 20 µg
Lane 2: Treated (10 ng/ml TNF-a+100 nM Smac mimetic+20 µM z-VAD 6 h) human hepatocyte cell lysate at 20 µg
All lanes: Goat anti-Rabbit HRP at 1/5000 dilution
Predicted band size: 54 kDa
Sample: HT-29 (Human colorectal adenocarcinoma epithelial cell) treated with 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 μM z-VAD for 8 hours whole cell lysates 10 μg per lane.
Lane 1 : Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091) at 0.12 μg/ml (Batch produced in 2016)
Lane 2 : Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091) at 0.17 μg/ml (Batch produced in 2015)
Lane 3 : Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091) at 0.12 μg/ml (GR212667 - batch produced in 2014)
Lane 4 : Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091) at 0.16 μg/ml (The supernatant of the clone producing ab187091)
Lane 5 : Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091) at 0.15 μg/ml (Batch produced in 2017)
Secondary
Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Blocking and diluting buffer: 5% NFDM/TBST.
The lysate in this image is prepared by 1%SDS Hot Lysate buffer.
For Lysate preparation protocol, please refer to the protocol section of the website and/or here.
All lanes: Western blot - Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091)
Predicted band size: 54 kDa
Observed band size: 54 kDa
Exposure time: 1min
Blocking and diluting buffer: 5% NFDM/TBST.
For 1%SDS Hot Lysate preparation protocol, please refer to the protocol section of the website and/or here.
All lanes: Western blot - Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091) at 1/5000 dilution
Lane 1: HT-29 (Human colorectal adenocarcinoma epithelial cell) treated with 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 µM z-VAD for 6 hr. The lysate is directly prepared by 1xSDS loading buffer. at 20 µg
Lane 2: HT-29 (Human colorectal adenocarcinoma epithelial cell) treated with 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 µM z-VAD for 8 hr. The lysate is prepared by 1%SDS Hot Lysate buffer method. at 20 µg
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Predicted band size: 54 kDa
Observed band size: 54 kDa
Exposure time: 3min
Details on WB tested positive control samples: HT-29 cells were treated with the indicated stimuli for 8 hr and then harvested. The final concentrations of 20 ng/ml TNF-a, 100 nM Smac mimetic, and 20 μM z-VAD were used to induce necrosis.
The lysate in this image is prepared by 1%SDS Hot Lysate buffer.
For Lysate preparation protocol, please refer to the protocol section of the website and/or here.
All lanes: Western blot - Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091) at 1/2000 dilution
Lane 1: Untreated HT-29 lysate at 10 µg
Lane 2: HT-29 cell lysate treated with TNF alpha+ Smac mimetic+ z-VAD at 10 µg
All lanes: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution
Predicted band size: 54 kDa
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