Recombinant
RabMAb

Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091)

Overview

  • Product name
    Anti-MLKL (phospho S358) antibody [EPR9514]
    See all MLKL primary antibodies
  • Description
    Rabbit monoclonal [EPR9514] to MLKL (phospho S358)
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, IHC-Pmore details
  • Species reactivity
    Reacts with: Human
    Does not react with: Mouse, Rat
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) within Human MLKL aa 350-450 (phospho S358). The exact sequence is proprietary.
    Database link: Q8NB16

  • Positive control
    • HT-29 cell lysate treated with TNF alpha+ Smac mimetic+ z-VAD. HT-29 cells were treated with the indicated stimuli for 8 hours and then harvested. The final concentrations of 20 ng/ml TNF-a, 100 nM Smac mimetic, and 20 µM z-VAD were used to induce necrosis
  • General notes

    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 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
    EPR9514
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab187091 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 1/1000 - 1/2000. Predicted molecular weight: 54 kDa.Can be blocked with Human MLKL (phospho S358) peptide (ab206929).
IHC-P 1/250 - 1/500.

Not Suitable for Mouse and Rat

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 S358) antibody [EPR9514] (ab187091) at 1/1000 dilution

    Lane 1 : Untreated HT-29 (human colorectal adenocarcinoma) whole cell lysates 20µg
    Lane 2 : HT-29 (human colorectal adenocarcinoma) treated with TNF alpha+ Smac mimetic+ z-VAD whole cell lysates 20µg
    Lane 3 : HT-29 (human colorectal adenocarcinoma) treated with TNF alpha+ Smac mimetic + z-VAD and phosphatase whole cell lysates 20µg.

    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



    Blocking buffer and concentration: 5% NFDM/TBST, Diluting buffer and concentration: 5% NFDM /TBST, Exposure time: 1 minute

    The lysate in this image is prepared by 1%SDS Hot Lysate buffer.

    For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).

  • ab187091 at 1:250 staining MLKL (phospho S358) in Human melanoma tissue by immunohistochemistry (FFPE).

     

    Antigen retrival required on FFPE tissue:  HIER using 10mM Citrate buffer pH 6.0, see recommended HIER protocol

     

    For additional IHC guideline, please see IHC resource page

  • ab187091 at 1:250 staining MLKL (phospho S358) in Human skin tissue by immunohistochemistry (FFPE).

     

    Antigen retrival required on FFPE tissue: HIER using 10mM Citrate buffer pH 6.0, see recommended HIER protocol

    For additional IHC guideline, please see IHC resource page

  • All lanes : Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091) at 1/5000 dilution

    Lane 1 : HT-29 (Human colorectal adenocarcinoma epithelial cell) treated with 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 µM z-VAD for 6 hr. The lysate is directly prepared by 1xSDS loading buffer.
    Lane 2 : HT-29 (Human colorectal adenocarcinoma epithelial cell) treated with 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 µM z-VAD for 8 hr. The lysate is prepared by 1%SDS Hot Lysate buffer method.

    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: 3 minutes


    Blocking and diluting buffer: 5% NFDM/TBST.

    For 1%SDS Hot Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).

  • Sample: HT-29 (Human colorectal adenocarcinoma epithelial cell) treated with 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 µM z-VAD for 8 hours whole cell lysates 10 µg per lane.
     
    Lane 1 : Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091) at 0.12 µg/ml (Batch produced in 2016)
    Lane 2 : Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091) at 0.17 µg/ml (Batch produced in 2015)
    Lane 3 : Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091) at 0.12 µg/ml (GR212667 - batch produced in 2014)
    Lane 4 : Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091) at 0.16 µg/ml (The supernatant of the clone producing ab187091)
    Lane 5 : Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091) at 0.15 µg/ml (Batch produced in 2017)
     
    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
     
     

    Blocking and diluting buffer: 5% NFDM/TBST.

    The lysate in this image is prepared by 1%SDS Hot Lysate buffer.

    For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).

  • All lanes : Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091) at 1/2000 dilution

    Lane 1 : Untreated HT-29 lysate
    Lane 2 : HT-29 cell lysate treated with TNF alpha+ Smac mimetic+ z-VAD

    Lysates/proteins at 10 µg per lane.

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

    Predicted band size: 54 kDa



    Details on WB tested positive control samples:  HT-29 cells were treated with the indicated stimuli for 8 hr and then harvested. The final concentrations of 20 ng/ml TNF-a, 100 nM Smac mimetic, and 20 µM z-VAD were used to induce necrosis.

    The lysate in this image is prepared by 1%SDS Hot Lysate buffer.

    For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).

  • Dot blot analysis of MLKL peptides using ab187091 at 1/1000 dilution followed by Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated secondary antibody at 1/1000 dilution. Blocking and diluting buffer was 5% NFDM/TBST, exposure time 3 minuts.

     

    Lane 1: MLKL (pT357) phospho peptide

    Lane 2: MLKL (pS358) phospho peptide

    Lane 3: MLKL (pT357/pS358) phospho peptide

    Lane 4: MLKL non-phospho peptide 

References

This product has been referenced in:

See all 35 Publications for this product

Customer reviews and Q&As

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunocytochemistry
Sample
Mouse Cultured Cells (Cardiomyocytes)
Specification
Cardiomyocytes
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Jan 31 2018

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Prostate cancer cell line)
Permeabilization
Yes - 0.1% TritonX-100
Specification
Prostate cancer cell line
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Methanol
Username

Miss. Pei-Fang Wu

Verified customer

Submitted Aug 07 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (ovary)
Gel Running Conditions
Reduced Denaturing (12)
Loading amount
7 µg
Treatment
50 ng/ml TNFa 24hrs
Specification
ovary
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Username

Konstantin Bagnjuk

Verified customer

Submitted Aug 04 2017

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Liver)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Ventana CC2 (pH6)
Permeabilization
No
Specification
Liver
Blocking step
Ventana blocking agent as blocking agent for 4 minute(s) · Concentration: 100% · Temperature: 36°C
Fixative
Formaldehyde
Username

Abcam user community

Verified customer

Submitted Feb 17 2017

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

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