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Rabbit Recombinant Monoclonal MLKL phospho S345 antibody. Carrier free. Suitable for IP, Dot, WB and reacts with Mouse, Synthetic peptide samples. Cited in 4 publications.

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Images

Immunoprecipitation - Anti-MLKL (phospho S345) antibody [EPR9515(2)] - BSA and Azide free (AB208910), expandable thumbnail
  • Dot Blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] - BSA and Azide free (AB208910), expandable thumbnail
  • Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] - BSA and Azide free (AB208910), expandable thumbnail
  • Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] - BSA and Azide free (AB208910), expandable thumbnail

Publications

Key facts

Isotype
IgG
Host species
Rabbit
Storage buffer

pH: 7.2 - 7.4
Constituents: PBS

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
Not recommended
Tested
Not recommended

Tested
Tested

Species
Mouse
Dilution info
-
Notes

-

Not recommended
Not recommended

Species
Synthetic peptide
Dilution info
-
Notes

-

Tested
Tested

Species
Synthetic peptide
Dilution info
-
Notes

-

Expected
Expected

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

-

Tested
Tested

Species
Mouse
Dilution info
-
Notes

-

Not recommended
Not recommended

Species
Synthetic peptide
Dilution info
-
Notes

-

Associated Products

Select an associated product type

9 products for Alternative Product

1 product for Alternative Version

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

Rabbit Recombinant Monoclonal MLKL phospho S345 antibody. Carrier free. Suitable for IP, Dot, WB and reacts with Mouse, Synthetic peptide samples. Cited in 4 publications.

Key facts

Isotype
IgG
Form
Liquid
Clonality
Monoclonal
Immunogen
  • The exact immunogen used to generate this antibody is proprietary information.
Carrier free
Yes
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 conditions
+4°C
Appropriate long-term storage conditions
+4°C
Storage information
Do Not Freeze

Notes

ab208910 is the carrier-free version of Anti-MLKL (phospho S345) antibody [EPR9515(2)] ab196436.

This antibody was developed through collaboration with the lab of Xiaodong Wang at the National Institute of Biological Sciences, Beijing.

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.

Our carrier-free antibodies are typically supplied in a PBS-only formulation, purified and free of BSA, sodium azide and glycerol. The carrier-free buffer and high concentration allow for increased conjugation efficiency.

This conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, which makes them ideal for antibody labelling, functional and cell-based assays, flow-based assays (e.g. mass cytometry) and Multiplex Imaging applications.

Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with 1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

This product is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm, without the need for antibody preparation. Maxpar® is a trademark of Fluidigm Canada Inc.

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.

Full details and terms and conditions can be found here:
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4 product images

  • Immunoprecipitation - Anti-MLKL (phospho S345) antibody [EPR9515(2)] - BSA and Azide free (ab208910), expandable thumbnail

    Immunoprecipitation - Anti-MLKL (phospho S345) antibody [EPR9515(2)] - BSA and Azide free (ab208910)

    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 + 100 nM Smac mimetic + 20 µM z-VAD compound for 8h using Anti-MLKL (phospho S345) antibody [EPR9515(2)] ab196436 at 1/150 dilution. Western blot was performed from the immunoprecipitate using Anti-MLKL (phospho S345) antibody [EPR9515(2)] 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+ 100 nM Smac mimetic+ 20 µM z-VAD compound for 8h;10 µg (Input).
    Lane 2: Anti-MLKL (phospho S345) antibody [EPR9515(2)] ab196436 IP in L-929 whole cell lysate treated with 20 ng/ml TNF alpha+ 100 nM Smac mimetic+ 20 µM z-VAD compound for 8h.
    Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of Anti-MLKL (phospho S345) antibody [EPR9515(2)] ab196436 in L-929 whole cell lysate treated with 20 ng/ml TNF alpha+ 100 nM Smac mimetic+ 20 µM z-VAD compound for 8h.

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

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-MLKL (phospho S345) antibody [EPR9515(2)] ab196436).

    All lanes: Immunoprecipitation - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (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)] - BSA and Azide free (ab208910), expandable thumbnail

    Dot Blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] - BSA and Azide free (ab208910)

    This Dot Blot data was generated using the same anti-MLKL (phospho S345) antibody clone, EPR9515(2), in a different buffer formulation (cat# 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 Anti-MLKL (phospho S345) antibody [EPR9515(2)] ab196436 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated secondary antibody at 1/1000 dilution.

    Blocking/Dilution buffer: 5% NFDM/TBST.

    Exposure time: 3 minutes.

  • Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] - BSA and Azide free (ab208910), expandable thumbnail

    Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] - BSA and Azide free (ab208910)

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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)] (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)] - BSA and Azide free (ab208910), expandable thumbnail

    Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] - BSA and Azide free (ab208910)

    This data was developed using Anti-MLKL (phospho S345) antibody [EPR9515(2)] ab196436, the same antibody clone in a different buffer formulation.

    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)] (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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