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Anti-MLKL (phospho S345) antibody [EPR9515(2)] is a rabbit recombinant monoclonal antibody that is used to detect MLKL in Dot, IP, Western blot. Suitable for Mouse samples.

- Using biophysical QC, Antibody identity is confirmed at a molecular level for unrivaled batch-batch consistency
- Antibody clone EPR9515(2) is the most widely used clone for MLKL on the market


Images

Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (AB196436), expandable thumbnail
  • Immunoprecipitation - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (AB196436), expandable thumbnail
  • Dot Blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (AB196436), expandable thumbnail
  • Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (AB196436), expandable thumbnail
  • Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (AB196436), expandable thumbnail

Publications

Key facts

Isotype
IgG
Host species
Rabbit
Storage buffer

pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA

Form
Liquid
Clonality
Monoclonal

Immunogen

  • The exact immunogen used to generate this antibody is proprietary information.

Reactivity data

Select an application
Product promiseTestedExpectedPredictedNot recommended
IPDotWB
Mouse
Tested
Expected
Tested
Synthetic peptide - Mouse
Not recommended
Tested
Not recommended

Tested
Tested

Species
Mouse
Dilution info
1/150
Notes

-

Not recommended
Not recommended

Species
Synthetic peptide - Mouse
Dilution info
-
Notes

-

Tested
Tested

Species
Synthetic peptide - Mouse
Dilution info
1/1000
Notes

-

Expected
Expected

Species
Mouse
Dilution info
Use at an assay dependent concentration.
Notes

-

Tested
Tested

Species
Mouse
Dilution info
1/1000
Notes

-

Not recommended
Not recommended

Species
Synthetic peptide - Mouse
Dilution info
-
Notes

-

Associated Products

Select an associated product type

11 products for Alternative Product

Target data

Function

Pseudokinase that plays a key role in TNF-induced necroptosis, a programmed cell death process (PubMed:23835476, PubMed:24012422, PubMed:24019532, PubMed:27321907, PubMed:32200799, PubMed:32296175). Does not have protein kinase activity (PubMed:24012422). 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:23835476, PubMed:24012422, PubMed:24019532, PubMed:27321907). In addition to TNF-induced necroptosis, necroptosis can also take place in the nucleus in response to orthomyxoviruses infection: 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 (PubMed:32200799, PubMed:32296175). Binds to highly phosphorylated inositol phosphates such as inositolhexakisphosphate (InsP6) which is essential for its necroptotic function (By similarity).

Alternative names

Mixed lineage kinase domain-like protein, Mlkl

Recommended products

Anti-MLKL (phospho S345) antibody [EPR9515(2)] is a rabbit recombinant monoclonal antibody that is used to detect MLKL in Dot, IP, Western blot. Suitable for Mouse samples.

- Using biophysical QC, Antibody identity is confirmed at a molecular level for unrivaled batch-batch consistency
- Antibody clone EPR9515(2) is the most widely used clone for MLKL on the market

Key facts

Isotype
IgG
Form
Liquid
Clonality
Monoclonal
Immunogen
  • The exact immunogen used to generate this antibody is proprietary information.
Clone number
EPR9515(2)
Purification technique
Affinity purification Protein A
Specificity

MLKL pS345 is a trigger for necroptosis. It is only detectable in infection/cellular damaged (PMID:29229989) or aging tissue (PMID: 28807105) but not in normal tissues.

Concentration
Loading...

Storage

Shipped at conditions
Blue Ice
Appropriate short-term storage duration
1-2 weeks
Appropriate short-term storage conditions
+4°C
Appropriate long-term storage conditions
-20°C
Aliquoting information
Upon delivery aliquot
Storage information
Avoid freeze / thaw cycle

Notes

Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436) was developed by Abcam using patented rabbit monoclonal antibody technology and is validated for use in Dot Blot, IP and WB.

Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436) was first used in a scientific publication in 2016 and has been cited over 288 times in peer reviewed journals. It's performance in Western Blot and IHC in mouse samples is trusted by the scientific community.

Abcam's high quality manufacturing and validation processes ensure Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436) has high sensitivity and specificity alongside high lot-to-lot consistency and reproducibility.

Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436) has 25 independent reviews from customers.

Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436) specifically detects MLKL Phospho-S345 (UniProt ID: Q9D2Y4; Molecular weight: 55kDa) and is sold in 100 µL and 1 mL selling sizes.

Conjugation-ready, carrier free format available for antibody clone EPR9515(2) - Anti-MLKL (phospho S345) antibody [EPR9515(2)] - BSA and Azide free ab208910.

Antibody clone EPR9515(2) is also available pre-conjugated to a variety of labels for your convenience - HRP (HRP Anti-MLKL (phospho S345) antibody [EPR9515(2)] ab208430).

Mixed lineage kinase domain-like protein (MLKL) is a key executor of necroptosis, a form of programmed cell death and its expression is associated with both tumor suppression and promotion, depending on the cancer type. Its role in necroptosis and other cellular processes makes it a potential prognostic marker and therapeutic target in oncology.

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:

  • - High batch-to-batch consistency and reproducibility
  • - Improved sensitivity and specificity
  • - Long-term security of supply
  • - Animal-free batch production

For more information, read more on recombinant antibodies.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.
Activity summary

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.

Biological function summary

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.

Pathways

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.

Associated diseases and disorders

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.

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.

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7 product images

  • Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436), expandable thumbnail

    Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436)

    Blocking and dilution buffer: 5% NFDM/TBST.

    All lanes: Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436) at 1/1000 dilution

    Lane 1: Untreated L-929 (Mouse connective tissue fibroblast cells) whole cell lysate at 10 µg

    Lane 2: L-929 whole cell lysate treated with 20 ng/ml TNF alpha (Recombinant human TNF alpha protein ab9642), 100 nM Smac mimetic, and 20 µM z-VAD (Z-VAD(OH)-FMK, Irreversible general caspase inhibitor ab120382) for 8 h and then harvested. at 10 µg

    Secondary

    All lanes: Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

    Predicted band size: 54 kDa

    Observed band size: 54 kDa

    Exposure time: 15s

  • Immunoprecipitation - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436), expandable thumbnail

    Immunoprecipitation - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436)

    MLKL (phospho S345) was immunoprecipitated from 1mg of L-929 (Mouse connective tissue fibroblast cells) whole cell lysate treated with 20 ng/ml TNF alpha (Recombinant human TNF alpha protein ab9642) + 100 nM Smac mimetic + 20 μM z-VAD compound (Z-VAD(OH)-FMK, Irreversible general caspase inhibitor ab120382) for 8h using ab196436 at 1/150 dilution. Western blot was performed from the immunoprecipitate using ab196436 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

    Lane 1: L-929 whole cell lysate treated with 20 ng/ml TNF alpha (Recombinant human TNF alpha protein ab9642) + 100 nM Smac mimetic+ 20 μM z-VAD compound (Z-VAD(OH)-FMK, Irreversible general caspase inhibitor ab120382) for 8h;10 μg (Input).
    Lane 2: ab196436 IP in L-929 whole cell lysate treated with 20 ng/ml TNF alpha (Recombinant human TNF alpha protein ab9642) + 100 nM Smac mimetic+ 20 μM z-VAD compound (Z-VAD(OH)-FMK, Irreversible general caspase inhibitor ab120382) for 8h.
    Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab196436 in L-929 whole cell lysate treated with 20 ng/ml TNF alpha (Recombinant human TNF alpha protein ab9642) + 100 nM Smac mimetic+ 20 μM z-VAD compound (Z-VAD(OH)-FMK, Irreversible general caspase inhibitor ab120382) for 8h.

    Blocking and dilution buffer and concentration: 5% NFDM/TBST.

    All lanes: Immunoprecipitation - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436)

    All lanes: Immunoprecipitation - Recombinant human TNF alpha protein (Recombinant human TNF alpha protein ab9642)

    Predicted band size: 54 kDa

    Observed band size: 54 kDa

  • Dot Blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436), expandable thumbnail

    Dot Blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436)

    Dot blot analysis of MLKL (phospho S345) peptide (Lane 1), and non-phospho peptide (Lane 2), labeled using ab196436 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated secondary antibody at 1/1000 dilution.

    Blocking and dilution buffer: 5% NFDM/TBST.

    Exposure time: 3 minutes.

  • Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436), expandable thumbnail

    Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436)

    Blocking and diluting buffer: 5% NFDM/TBST.

    MLKL pS345 is a trigger for necroptosis. It is only detectable in infection/cellular damaged (PMID:29229989) or aging tissue (PMID: 28807105) but not in normal tissues.

    All lanes: Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436) at 1/1000 dilution

    Lane 1: L-929 treated with 20 ng/ml TNF alpha (Recombinant human TNF alpha protein ab9642), 100 nM Smac mimetic, and 20 µM z-VAD (Z-VAD(OH)-FMK, Irreversible general caspase inhibitor ab120382) for 8 h, whole cell lysate at 20 µg

    Lane 2: Mouse brain tissue lysate at 20 µg

    Lane 3: Mouse colon tissue lysate at 20 µg

    Lane 4: Mouse lung tissue lysate at 20 µg

    Lane 5: Mouse retina tissue lysate at 20 µg

    Lane 6: Mouse liver tissue lysate at 20 µg

    Lane 7: Raw264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

    Secondary

    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: 50s

  • Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436), expandable thumbnail

    Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436)

    Blocking and diluting buffer: 5% NFDM/TBTS.

    Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 was used as a loading control at 1/1000000 dilution.

    We recommend involving downstream protein p-MLKL as a control to validate the stimulation of p-RIP3.

    Lanes 1 - 4: Western blot - Anti-RIP3 (phospho T231 + S232) antibody [2D7] (Anti-RIP3 (phospho T231 + S232) antibody [2D7] ab205421) at 1/1000 dilution

    Lanes 5 - 8: Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436) at 1/1000 dilution

    Lanes 1, 3, 5 and 7: Untreated L-929 (mouse connective tissue fibroblast cell line) whole cell lysate at 20 µg

    Lanes 2 and 6: L-929 (mouse connective tissue fibroblast cell line) treated with 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 μM z-VAD for 8 hours whole cell lysate at 20 µg

    Lanes 4 and 8: L-929 (mouse connective tissue fibroblast cell line) treated with 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 μM z-VAD for 6 hours whole cell lysate at 20 µg

    Secondary

    Lanes 1 - 4: Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/20000 dilution

    Lanes 5 - 8: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

    Predicted band size: 57 kDa, 54 kDa

    Observed band size: 53 kDa

    Exposure time: 180s

  • Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436), expandable thumbnail

    Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436)

    Blocking and diluting buffer: 5% NFDM/TBST.

    Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 was used as a loading control at 1/1000000 dilution.

    We recommend involving downstream protein p-MLKL as a control to validate the stimulation of p-RIP3.

    Lanes 1 - 4: Western blot - Anti-RIP3 (phospho S232) antibody [EPR9516(N)-25] (Anti-RIP3 (phospho S232) antibody [EPR9516(N)-25] ab195117) at 1/1000 dilution

    Lanes 5 - 8: Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436) at 1/1000 dilution

    Lanes 1, 3, 5 and 7: Untreated L-929 (mouse connective tissue fibroblast cell line) whole cell lysate at 20 µg

    Lanes 2 and 6: L-929 (mouse connective tissue fibroblast cell line) treated with 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 μM z-VAD for 8 hours whole cell lysate at 20 µg

    Lanes 4 and 8: L-929 (mouse connective tissue fibroblast cell line) treated with 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 μM z-VAD for 6 hours whole cell lysate at 20 µg

    Secondary

    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: 57 kDa, 54 kDa

    Observed band size: 53 kDa

    Exposure time: 180s

  • Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436), expandable thumbnail

    Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436)

    Blocking and diluting buffer concentration: 5% NFDM/TBST.

    Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 GAPDH was used as a loading control at 1/1000000 dilution.

    We recommend involving downstream protein p-MLKL as a control to validate the stimulation of p-RIP3.

    Lanes 1 - 4: Western blot - Anti-RIP3 (phospho T231 + S232) antibody [EPR19403-52] (Anti-RIP3 (phospho T231 + S232) antibody [EPR19403-52] ab222320) at 1/1000 dilution

    Lanes 5 - 8: Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436) at 1/1000 dilution

    Lanes 1, 3, 5 and 7: Untreated L-929 (mouse connective tissue fibroblast cell line) whole cell lysate at 20 µg

    Lanes 2 and 6: L-929 (mouse connective tissue fibroblast cell line) treated with 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 μM z-VAD for 8 hours whole cell lysate at 20 µg

    Lanes 4 and 8: L-929 (mouse connective tissue fibroblast cell line) treated with 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 μM z-VAD for 6 hours whole cell lysate at 20 µg

    Secondary

    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: 57 kDa, 54 kDa

    Observed band size: 53 kDa

    Exposure time: 180s

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Product protocols

For this product, it's our understanding that no specific protocols are required. You can:

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