Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-MLKL antibody [EPR17514] (ab184718)

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Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR17514] to MLKL
  • Suitable for: ICC/IF, WB, IHC-P
  • Knockout validated
  • Reacts with: Human

Overview

  • Product name

    Anti-MLKL antibody [EPR17514]
    See all MLKL primary antibodies

  • Description

    Rabbit monoclonal [EPR17514] to MLKL

  • Host species

    Rabbit

  • Tested applications

    Suitable for: ICC/IF, WB, IHC-Pmore details

  • Species reactivity

    Reacts with: Human

  • Immunogen

    Recombinant fragment within Human MLKL aa 350 to the C-terminus. The exact sequence is proprietary.
    Database link: Q8NB16

  • Positive control

    • WB: HUVEC, HT-29 and HeLa whole cell lysates; Human fetal kidney lysate. IHC-P: Human tonsil and colonic adenocarcinoma tissues.

  • General notes

    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 production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

Properties

  • Form

    Liquid

  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.

  • Storage buffer

    Preservative: 0.01% Sodium azide
    Constituents: 59% PBS, 40% Glycerol, 0.05% BSA
  • Concentration information loading...

  • Purity

    Protein A purified

  • Clonality

    Monoclonal

  • Clone number

    EPR17514

  • Isotype

    IgG

  • Research areas

Applications

Our Abpromise guarantee covers the use of ab184718 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ICC/IF 1/200.
WB 1/1000. Detects a band of approximately 54 kDa (predicted molecular weight: 54 kDa).
IHC-P 1/400. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Target

  • Sequence similarities

    Belongs to the protein kinase superfamily.
    Contains 1 protein kinase domain.

  • Domain

    The protein kinase domain is predicted to be catalytically inactive.
  • Information by UniProt

  • Database links

    • Alternative names

      • 9130019I15Rik antibody
      • FLJ34389 antibody
      • hMLKL antibody
      • Mixed lineage kinase domain like antibody
      • Mixed lineage kinase domain like protein antibody
      • Mixed lineage kinase domain like pseudokinase antibody
      • Mixed lineage kinase domain-like protein antibody
      • Mlkl antibody
      • MLKL_HUMAN antibody
      see all

    Images

    • Western blot - Anti-MLKL antibody [EPR17514] (ab184718)
      Western blot - Anti-MLKL antibody [EPR17514] (ab184718)
      All lanes : Anti-MLKL antibody [EPR17514] (ab184718) at 1/1000 dilution

      Lane 1 : HUVEC cell lysate
      Lane 2 : HT-29 cell lysate
      Lane 3 : Wild-type HeLa cell lysate
      Lane 4 : MLKL knockout HeLa cell lysate

      Lysates/proteins at 20 µg per lane.

      Predicted band size: 54 kDa



      Lanes 1 - 4: Merged signal (red and green). Green - ab184718 observed at 54 kDa. Red - loading control, ab8245 observed at 37 kDa.

       ab184718 was shown to react with MLKL in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab255408 (knockout cell lysate ab263788) was used. Wild-type and MLKL knockout samples were subjected to SDS-PAGE. ab184718 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

       

    • Western blot - Anti-MLKL antibody [EPR17514] (ab184718)
      Western blot - Anti-MLKL antibody [EPR17514] (ab184718)
      All lanes : Anti-MLKL antibody [EPR17514] (ab184718) at 1/1000 dilution

      Lane 1 : Wild-type HAP1 cell lysate
      Lane 2 : MLKL knockout HAP1 cell lysate
      Lane 3 : HeLa cell lysate
      Lane 4 : Huvec cell lysate

      Lysates/proteins at 20 µg per lane.

      Predicted band size: 54 kDa



      Lanes 1 - 4: Merged signal (red and green). Green - ab184718 observed at 55 kDa. Red - loading control, ab8245, observed at 37 kDa.


      ab184718 was shown to recognize MLKL in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when MLKL knockout samples were examined. Wild-type and MLKL knockout samples were subjected to SDS-PAGE. ab184718 and ab8245 (loading control to GAPDH) were diluted to 1/1000 and 1/2000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

    • Immunocytochemistry/ Immunofluorescence - Anti-MLKL antibody [EPR17514] (ab184718)
      Immunocytochemistry/ Immunofluorescence - Anti-MLKL antibody [EPR17514] (ab184718)

      Ab184718 staining MLKL in HT-29 (Human colorectal adenocarcinoma epithelial cell) cells by Immunocytochemistry (ICC). Cells were fixed with 100% Methanol. Samples were incubated with primary antibody at 1/200 dilution (6.5μg/ml).  An AlexaFluor® 488 Goat anti-Rabbit (ab150077) was used as the secondary antibody at 1/1000 dilution (2μg/ml). Ab195889 , Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594)  was used as the counterstain antibody (1/200 dilution, 2.5 μg/ml . DAPI was used as a nuclear counterstain. Confocal image showing cytoplasmic staining on HT-29 cell line.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MLKL antibody [EPR17514] (ab184718)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MLKL antibody [EPR17514] (ab184718)

      Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling MLKL with ab184718 at 1/400 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) secondary antibody (ab97051) at 1/500 dilution. Cytoplasmic staining on the lymphocytes of human tonsil is observed. Counter stained with Hematoxylin.

      Negative control: Using PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

      Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MLKL antibody [EPR17514] (ab184718)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MLKL antibody [EPR17514] (ab184718)

      Immunohistochemical analysis of paraffin-embedded Human colonic adenocarcinoma tissue labeling MLKL with ab184718 at 1/400 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) secondary antibody (ab97051) at 1/500 dilution. Cytoplasmic staining on tumor cells of human colonic adenocarcinoma is observed. Counter stained with Hematoxylin.

      Negative control: Using PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

      Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

    • Western blot - Anti-MLKL antibody [EPR17514] (ab184718)
      Western blot - Anti-MLKL antibody [EPR17514] (ab184718)
      All lanes : Anti-MLKL antibody [EPR17514] (ab184718) at 1/20000 dilution

      Lane 1 : HUVEC (Human umbilical vein endothelial cell line) whole cell lysate
      Lane 2 : HT-29 (Human colorectal adenocarcinoma cells) whole cell lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

      Predicted band size: 54 kDa



      Blocking/Dilution buffer: 5% NFDM/TBST.

    • Western blot - Anti-MLKL antibody [EPR17514] (ab184718)
      Western blot - Anti-MLKL antibody [EPR17514] (ab184718)
      Anti-MLKL antibody [EPR17514] (ab184718) at 1/1000 dilution + Human fetal kidney lysate at 10 µg

      Secondary
      Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

      Predicted band size: 54 kDa
      Observed band size: 54 kDa



      Blocking/Dilution buffer: 5% NFDM/TBST.

    References

    Publishing research using ab184718? Please let us know so that we can cite the reference in this datasheet.

    ab184718 has been referenced in 20 publications.

    • Zhang J  et al. Membrane-bound TNF mediates microtubule-targeting chemotherapeutics-induced cancer cytolysis via juxtacrine inter-cancer-cell death signaling. Cell Death Differ 27:1569-1587 (2020). PubMed: 31645676
    • Kant S  et al. Enhanced fatty acid oxidation provides glioblastoma cells metabolic plasticity to accommodate to its dynamic nutrient microenvironment. Cell Death Dis 11:253 (2020). PubMed: 32312953
    • Wang Y  et al. Necroptosis regulates tumor repopulation after radiotherapy via RIP1/RIP3/MLKL/JNK/IL8 pathway. J Exp Clin Cancer Res 38:461 (2019). PubMed: 31706322
    • Li J  et al. Transcription modulation by CDK9 regulates inflammatory genes and RIPK3-MLKL-mediated necroptosis in periodontitis progression. Sci Rep 9:17369 (2019). PubMed: 31758083
    • Xie Y  et al. Phosphorylated mixed lineage kinase domain-like protein in human seminal plasma: A potential novel biomarker of spermatogenic function. Andrologia 51:e13310 (2019). PubMed: 31095773
    View all Publications for this product

    Customer reviews and Q&As

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    1-4 of 4 Abreviews or Q&A

    Western blot abreview for Anti-MLKL antibody [EPR17514]

     Excellent
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (ovary)
    Gel Running Conditions
    Reduced Denaturing (12)
    Loading amount
    7 µg
    Specification
    ovary
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
    Read More

    Konstantin Bagnjuk

    Verified customer

    Submitted Aug 04 2017

    Western blot abreview for Anti-MLKL antibody [EPR17514]

     Excellent
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HeLa, HT29, L929, MEF)
    Gel Running Conditions
    Reduced Denaturing (10%)
    Loading amount
    10 µg
    Specification
    HeLa, HT29, L929, MEF
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
    Read More

    Xiaozheng Xu

    Verified customer

    Submitted Sep 08 2015

    Immunocytochemistry/ Immunofluorescence abreview for Anti-MLKL antibody [EPR17514]

     Excellent
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (HeLa)
    Permeabilization
    Yes - Triton X-100 0.25%
    Specification
    HeLa
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 22°C
    Fixative
    Paraformaldehyde
    Read More

    Xiaozheng Xu

    Verified customer

    Submitted Sep 07 2015

    Western blot abreview for Anti-MLKL antibody [EPR17514]

     Excellent
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HeLa Cell)
    Gel Running Conditions
    Reduced Denaturing
    Loading amount
    20 µg
    Specification
    HeLa Cell
    Read More

    Abcam user community

    Verified customer

    Submitted Apr 24 2015

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