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  1. Link

    mlkl-phospho-s345-antibody-epr95152-ab196436.pdf

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Signal Transduction Protein Phosphorylation Ser / Thr Kinases Other Kinases
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RecombinantRabMAb

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

  • Datasheet
  • SDS
  • Certificate of Compliance
Reviews (25) Submit a question References (193)

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Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436)
  • Immunoprecipitation - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436)
  • Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436)
  • Dot Blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436)
  • Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR9515(2)] to MLKL (phospho S345)
  • Suitable for: WB, IP, Dot blot
  • Reacts with: Mouse

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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: WB, IP, Dot blotmore details
  • Species reactivity

    Reacts with: Mouse
  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • 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.
  • General notes

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

    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

    pH: 7.2
    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

    • Signal Transduction
    • Protein Phosphorylation
    • Ser / Thr Kinases
    • Other Kinases
    • Microbiology
    • Organism
    • Virus
    • RNA Virus
    • ssRNA positive strand virus
    • SARS Coronavirus
    • Cancer
    • Cell Death
    • Necroptosis

Associated products

  • Alternative Versions

    • HRP Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab208430)
    • Anti-MLKL (phospho S345) antibody [EPR9515(2)] - BSA and Azide free (ab208910)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • Recombinant Protein

    • Recombinant Human MLKL protein (ab153623)
  • Related Products

    • Prestained Protein Ladder – Broad molecular weight (10-245 kDa) (ab116028)
    • Z-VAD(OH)-FMK, Irreversible general caspase inhibitor (ab120382)
    • Recombinant human TNF alpha protein (ab9642)

Applications

The Abpromise guarantee

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
WB (12)
1/1000. Detects a band of approximately 54 kDa (predicted molecular weight: 54 kDa).
IP (1)
1/150.
Dot blot
1/1000.
Notes
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.
  • Target information above from: UniProt accession Q8NB16 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 74568 Mouse
    • SwissProt: Q9D2Y4 Mouse
    • Unigene: 207971 Mouse
    • 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 (phospho S345) antibody [EPR9515(2)] (ab196436)
      Western blot - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436)
      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.

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

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

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

    Protocols

    • Western blot protocols
    • Immunoprecipitation protocols

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    Certificate of Compliance

    To download a Certificate of Compliance, please enter your Lot number below:

    References (193)

    Publishing research using ab196436? Please let us know so that we can cite the reference in this datasheet.

    ab196436 has been referenced in 193 publications.

    • Kist M  et al. Impaired RIPK1 ubiquitination sensitizes mice to TNF toxicity and inflammatory cell death. Cell Death Differ 28:985-1000 (2021). PubMed: 32999468
    • Salvany S  et al. Microglial recruitment and mechanisms involved in the disruption of afferent synaptic terminals on spinal cord motor neurons after acute peripheral nerve injury. Glia 69:1216-1240 (2021). PubMed: 33386754
    • Chen C  et al. Legumain promotes tubular ferroptosis by facilitating chaperone-mediated autophagy of GPX4 in AKI. Cell Death Dis 12:65 (2021). PubMed: 33431801
    • He P  et al. Detection of necroptosis by phospho-MLKL immunohistochemical labeling. STAR Protoc 2:100251 (2021). PubMed: 33458710
    • Wang P  et al. Essential role for autophagy protein VMP1 in maintaining neuronal homeostasis and preventing axonal degeneration. Cell Death Dis 12:116 (2021). PubMed: 33483473
    View all Publications for this product

    Customer reviews and Q&As

    Show All Reviews Q&A
    Submit a review Submit a question

    1-10 of 25 Abreviews or Q&A

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

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Mouse Tissue lysate - whole (BMDM)
    Gel Running Conditions
    Reduced Denaturing (8%)
    Loading amount
    20 µg
    Treatment
    LPS+Z-vad for 3 and 6 hours
    Specification
    BMDM
    Blocking step
    Milk as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: 25°C
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Mr. Yuanxin Yang

    Verified customer

    Submitted Feb 02 2021

    Immunocytochemistry/ Immunofluorescence abreview for Anti-MLKL (phospho S345) antibody [EPR9515(2)]

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Mouse Cell (liver)
    Permeabilization
    No
    Specification
    liver
    Blocking step
    Ventanas blocking kit as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 37°C
    Fixative
    Formaldehyde
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Nov 27 2020

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-MLKL (phospho S345) antibody [EPR9515(2)]

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Mouse Tissue sections (Spleen)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: Venta cell conditioner
    Permeabilization
    No
    Specification
    Spleen
    Blocking step
    Ventanas blocking kit as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 37°C
    Fixative
    Formaldehyde
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Nov 27 2020

    IHC - Wholemount abreview for Anti-MLKL (phospho S345) antibody [EPR9515(2)]

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    IHC - Wholemount
    Sample
    Mouse Tissue (Brain isolated capillaries)
    Specification
    Brain isolated capillaries
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Sep 19 2019

    Immunohistochemistry free floating abreview for Anti-MLKL (phospho S345) antibody [EPR9515(2)]

    Average
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry free floating
    Sample
    Mouse Tissue sections (Mouse brain free-floating section)
    Specification
    Mouse brain free-floating section
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Nov 24 2021

    Immunohistochemistry (Frozen sections) abreview for Anti-MLKL (phospho S345) antibody [EPR9515(2)]

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Frozen sections)
    Sample
    Mouse Tissue sections (Spleen)
    Permeabilization
    No
    Specification
    Spleen
    Blocking step
    Ventanas blocking kit as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 37°C
    Fixative
    Formaldehyde
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Nov 27 2020

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-MLKL (phospho S345) antibody [EPR9515(2)]

    Inconclusive
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Mouse Tissue sections (Embryo)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: Citrate buffer, pH 6
    Permeabilization
    Yes - 0.3% Triton X-100
    Specification
    Embryo
    Fixative
    Paraformaldehyde
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Jul 14 2020

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

    Average
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Mouse Tissue lysate - whole (heart)
    Gel Running Conditions
    Non-reduced Denaturing (10%)
    Loading amount
    30 µg
    Specification
    heart
    Blocking step
    (agent) for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Jun 24 2020

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-MLKL (phospho S345) antibody [EPR9515(2)]

    Good
    Abreviews
    Abreviews
    abreview image
    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
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Aug 01 2018

    Immunohistochemistry (PFA perfusion fixed frozen sections) abreview for Anti-MLKL (phospho S345) antibody [EPR9515(2)]

    Poor
    Abreviews
    Abreviews
    abreview image
    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
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    MRS. Francoise Geffroy

    Verified customer

    Submitted Mar 05 2018

    1-10 of 25 Abreviews or Q&A

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