Overview

  • Product name
    Anti-MLKL (phospho S345) antibody [EPR9515(2)]
    See all MLKL primary antibodies
  • Description
    Rabbit monoclonal [EPR9515(2)] to MLKL (phospho S345)
  • Host species
    Rabbit
  • 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.
  • Tested applications
    Suitable for: ICC/IF, WB, IP, Dot blotmore details
  • Species reactivity
    Reacts with: Mouse
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) within Mouse MLKL aa 300-400 (phospho S345). The exact sequence is proprietary.
    Database link: Q9D2Y4

  • Positive control
    • WB: L929 whole cell lysate treated with 20 ng/ml TNF alpha (ab9642), 100 nM Smac mimetic, and 20 µM z-VAD (ab120382) for 8h and then harvested. IP: L929 whole cell lysate treated with TNF alpha (ab9642) + Smac mimetic + z-VAD (ab120382) compound for 8h. ICC/IF: Mouse cardiomyocytes.
  • General notes

    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 (ab205718) and Goat Anti-Rabbit Alexa Fluor® 488 (ab150077).

    See other anti-rabbit secondary antibodies that can be used with this antibody.

    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 rabbit monoclonal antibody.

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
    EPR9515(2)
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab196436 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 1/100.
WB 1/1000. Detects a band of approximately 54 kDa (predicted molecular weight: 54 kDa).
IP 1/150.
Dot blot 1/1000.

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

  • All lanes : 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
    Lane 2 : L-929 whole cell lysate treated with 20 ng/ml TNF alpha (ab9642), 100 nM Smac mimetic, and 20 µM z-VAD (ab120382) for 8 h and then harvested.

    Lysates/proteins at 10 µg per lane.

    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: 15 seconds


    Blocking and dilution buffer: 5% NFDM/TBST.

  • Ab196436 staining MLKL in mouse cardiomyocytes by Immunocytochemistry. Samples were fixed with paraformaldehyde, permeabilized with 0.03% Triton x-100 and blocked with 1% BSA for 1 hour at room temperature. Samples were incubated with  primary antibody at 1/100 dilution for 12 hours. An Alexa Fluor® anti- rabbit was used as a secondary antibody at 1/200 dilution. 

    See Abreview

  • 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 (ab9642) + 100 nM Smac mimetic + 20 µM z-VAD compound (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 (ab9642) + 100 nM Smac mimetic+ 20 µM z-VAD compound (ab120382) for 8h;10 µg (Input).
    Lane 2: ab196436 IP in L-929 whole cell lysate treated with 20 ng/ml TNF alpha (ab9642) + 100 nM Smac mimetic+ 20 µM z-VAD compound (ab120382) for 8h.
    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab196436 in L-929 whole cell lysate treated with 20 ng/ml TNF alpha (ab9642) + 100 nM Smac mimetic+ 20 µM z-VAD compound (ab120382) for 8h.

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

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

    Lane 1 : L-929 treated with 20 ng/ml TNF alpha (ab9642), 100 nM Smac mimetic, and 20 µM z-VAD (ab120382) for 8 h, whole cell lysate
    Lane 2 : Mouse brain tissue lysate
    Lane 3 : Mouse colon tissue lysate
    Lane 4 : Mouse lung tissue lysate
    Lane 5 : Mouse retina tissue lysate
    Lane 6 : Mouse liver tissue lysate
    Lane 7 : Raw264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

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


    Exposure time: 50 seconds


    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.

  • 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.

References

This product has been referenced in:
  • Yuan Z  et al. A new perspective of triptolide-associated hepatotoxicity: Liver hypersensitivity upon LPS stimulation. Toxicology 414:45-56 (2019). Read more (PubMed: 30633930) »
  • Frank D  et al. Activated MLKL attenuates autophagy following its translocation to intracellular membranes. J Cell Sci 132:N/A (2019). Read more (PubMed: 30709919) »
See all 40 Publications for this product

Customer reviews and Q&As

1-10 of 16 Abreviews or Q&A

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Rat Tissue sections (brain)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Sodium citrate
Permeabilization
No
Specification
brain
Blocking step
BSA as blocking agent for 15 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Aug 01 2018

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample
Mouse Tissue sections (brain)
Antigen retrieval step
Heat mediated
Permeabilization
Yes - triton 0.2%
Specification
brain
Blocking step
Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5 · Temperature: 20°C
Fixative
Paraformaldehyde

Mrs. Francoise Geffroy

Verified customer

Submitted Mar 05 2018

Application
Immunocytochemistry
Sample
Mouse Cultured Cells (Cardiomyocytes)
Permeabilization
Yes - Triton x-100, 0.03%
Specification
Cardiomyocytes
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Jan 31 2018

Abcam has not validated the combination of species/application used in this Abreview.
Application
Western blot
Sample
Chinese hamster Cell lysate - whole cell (Chinese hamster ovary (CHO) cells)
Gel Running Conditions
Reduced Denaturing (4-12% Bolt Bis-Tris Plus gel (MES buffer))
Loading amount
50000 cells
Specification
Chinese hamster ovary (CHO) cells
Blocking step
Li-Cor Odyssey Blocking Buffer (TBS) as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Dec 31 2016

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Rat Cell (primary hippocampal neurons treated with TNF-a/ZVA)
Permeabilization
Yes - triton X-100
Specification
primary hippocampal neurons treated with TNF-a/ZVA
Blocking step
Gelatin from cold water fish skin as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Dec 20 2016

Application
Western blot
Sample
Rabbit Cell lysate - whole cell (Rabbit Reticulocyte)
Gel Running Conditions
Reduced Denaturing (4-12% Bolt Bis-Tris Plus gel (MES buffer))
Loading amount
0.5 µg
Specification
Rabbit Reticulocyte
Blocking step
Li-Cor Odyssey Blocking Buffer (TBS) as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Dec 12 2016

Application
Western blot
Sample
Rat Tissue lysate - whole (Brain)
Gel Running Conditions
Reduced Denaturing (10%)
Loading amount
40 µg
Specification
Brain
Blocking step
Milk as blocking agent for 40 minute(s) · Concentration: 5% · Temperature: 22°C

Abcam user community

Verified customer

Submitted Oct 27 2016

Application
Western blot
Sample
Rat Tissue lysate - whole (thymus)
Gel Running Conditions
Reduced Denaturing (4-15% BioRad TGX gel)
Loading amount
15 µg
Treatment
saline (CT) vs. methamphetamine (MT) 25mg/kg i.p., 6hr survival
Specification
thymus
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Abcam user community

Verified customer

Submitted Sep 05 2016

Application
Western blot
Sample
Human Cell lysate - whole cell (A549 (human lung epithelium cells))
Gel Running Conditions
Reduced Denaturing (4-12% Bolt Bis-Tris Plus gel)
Loading amount
50000 cells
Treatment
TNFalpha (25ng/mL) and zVAD (50uM) for 5 hours
Specification
A549 (human lung epithelium cells)
Blocking step
Li-Cor Odyssey Blocking Buffer (TBS) as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Aug 10 2016

Application
Western blot
Sample
African Green Monkey Cell lysate - whole cell (VERO E6)
Gel Running Conditions
Reduced Denaturing (4-12% Bolt Bis-Tris Plus gel)
Loading amount
75000 cells
Treatment
TNFalpha (25ng/mL) and zVAD (50uM) for 5 hours
Specification
VERO E6
Blocking step
Li-Cor Odyssey Blocking Buffer (TBS) as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Aug 09 2016

1-10 of 16 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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