Astana Biomed Group, an authorized Abcam distributor in Central Asia

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

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR9514] to MLKL (phospho S358)
  • Suitable for: WB, Dot blot, IHC-P
  • Reacts with: Human

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

  • Specificity

    Stimulation may be required to allow detection of the phosphorylated protein. Please see images below for recommended treatment conditions and positive controls.

  • Tested Applications & Species

    Application Species
    IHC-P
    Human
    WB
    Human
    See all applications and species data

  • Immunogen

    This information is proprietary to Abcam and/or its suppliers.
    (Peptide available as ab206929)

  • Positive control

    • WB: 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; human hepatocyte (treated with Smac/z0VAD) cell lysate. IHC-P: Human skin and melanoma tissue. Dot blot: MLKL (pS358) phospho peptide; MLKL (pT357/pS358) phospho peptide.

  • 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.

    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

    EPR9514

  • Isotype

    IgG

  • Research areas

Images

  • Western blot - Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091)
    Western blot - Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091)
    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).

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091)

    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

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091)

    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

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

    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 

  • Western blot - Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091)
    Western blot - Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091) Data courtesy of an anonymous AbReview
    All lanes : Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091) at 1000 cells

    Lane 1 : Untreated human hepatocyte cell lysate
    Lane 2 : Treated (10 ng/ml TNF-a+100 nM Smac mimetic+20 µM z-VAD 6 h) human hepatocyte cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat anti-Rabbit HRP at 1/5000 dilution

    Predicted band size: 54 kDa

    See Abreview

  • Western blot - Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091)
    Western blot - Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091)
    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).

  • Western blot - Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091)
    Western blot - Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091)
    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).

  • Western blot - Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091)
    Western blot - Anti-MLKL (phospho S358) antibody [EPR9514] (ab187091)
    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).

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

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