Name: Anti-MLKL (phospho S358) antibody [EPR9514]
Brand: RabMAb
SKU: ab187091
Rating: 3 (13 reviews)

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Western blot - 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

Carrier Free

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

Suitable for: WB, Dot blot, IHC-Pmore details
Species reactivity

Reacts with: Human
Does not react with: Mouse, Rat
Immunogen

Synthetic peptide. 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.

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

Alternative Versions
- Anti-MLKL (phospho S358) antibody [EPR9514] - BSA and Azide free (ab208909)
Compatible Secondaries
- Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
Immunizing Peptide (Blocking)
- Human MLKL (phospho S358) peptide (ab206929)
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)

The Abpromise guarantee

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	(7)	1/1000 - 1/2000. Predicted molecular weight: 54 kDa.Can be blocked with Human MLKL (phospho S358) peptide (ab206929). We recommend using 1% SDS Hot lysis method to prepare cell lysates. For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).
Dot blot		1/1000.
IHC-P	(2)	1/250 - 1/500. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. Not Suitable for Mouse and Rat

Notes
WB 1/1000 - 1/2000. Predicted molecular weight: 54 kDa.Can be blocked with Human MLKL (phospho S358) peptide (ab206929). We recommend using 1% SDS Hot lysis method to prepare cell lysates. For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).
Dot blot 1/1000.
IHC-P 1/250 - 1/500. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. Not Suitable for Mouse and Rat

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: 197259 Human
- Omim: 615153 Human
- SwissProt: Q8NB16 Human
- Unigene: 119878 Human
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

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)

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)

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 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)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)

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)

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)

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)

Western blot protocols
Immunohistochemistry protocols
Protocol Booklet

Click here to view the general protocols

SDS download

Country/Region

Language
Datasheet download

Download

Publishing research using ab187091? Please let us know so that we can cite the reference in this datasheet.

ab187091 has been referenced in 131 publications.

Ji Y et al. Reconstitution of Human Necrosome Interactions in Saccharomyces cerevisiae. Biomolecules 11:N/A (2021). PubMed: 33503908
Wang KJ et al. Emodin Induced Necroptosis and Inhibited Glycolysis in the Renal Cancer Cells by Enhancing ROS. Oxid Med Cell Longev 2021:8840590 (2021). PubMed: 33532038
Cai J et al. ABIN-1 is a key regulator in RIPK1-dependent apoptosis (RDA) and necroptosis, and ABIN-1 deficiency potentiates necroptosis-based cancer therapy in colorectal cancer. Cell Death Dis 12:140 (2021). PubMed: 33542218
Liu B et al. Sodium iodate induces ferroptosis in human retinal pigment epithelium ARPE-19 cells. Cell Death Dis 12:230 (2021). PubMed: 33658488
Hildebrand JM et al. A family harboring an MLKL loss of function variant implicates impaired necroptosis in diabetes. Cell Death Dis 12:345 (2021). PubMed: 33795639

View all Publications for this product

Customer reviews and Q&As

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1-10 of 13 Abreviews or Q&A

Application

Western blot

Sample

Human Cell lysate - whole cell (HT-29)

Gel Running Conditions

Reduced Denaturing (8%)

Loading amount

20 µg

Treatment

1µM GNF-7 for 24hrs

Specification

HT-29

Blocking step

BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Dr. Naghmeh Abiri

Verified customer

Submitted Mar 03 2021

Application

Immunocytochemistry/ Immunofluorescence

Sample

Rat Cell (HeLa cells)

Permeabilization

Yes - 0.1% Triton X-100

Specification

HeLa cells

Blocking step

BSA as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: 37°C

Fixative

Formaldehyde

Abcam user community

Verified customer

Submitted Dec 03 2020

Application

Immunohistochemistry (Frozen sections)

Sample

Rat Tissue sections (lung)

Permeabilization

Specification

lung

Blocking step

Ventanas blocking kit as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 37°C

Fixative

Formaldehyde

Abcam user community

Verified customer

Submitted Dec 03 2020

Application

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)

Sample

Rat Tissue sections (Spleen)

Antigen retrieval step

Heat mediated - Buffer/Enzyme Used: Venta cell conditioner

Permeabilization

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

Abcam user community

Verified customer

Submitted Dec 03 2020

Application

Western blot

Sample

Human Cell lysate - whole cell (Dopaminergic Neurons)

Gel Running Conditions

Reduced Denaturing

Loading amount

30 µg

Specification

Dopaminergic Neurons

Blocking step

BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Oct 22 2020

Application

Western blot

Sample

Human Cell lysate - whole cell (colon cancer)

Gel Running Conditions

Reduced Denaturing

Loading amount

30 µg

Specification

colon cancer

Blocking step

Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Oct 13 2020

Application

Western blot

Sample

Dog Cell lysate - whole cell (melanoma cell line)

Gel Running Conditions

Reduced Denaturing

Loading amount

10 µg

Specification

melanoma cell line

Blocking step

block ace as blocking agent for 50 minute(s) · Concentration: 100% · Temperature: 25°C

DR. 令中野

Verified customer

Submitted Jun 17 2019

Application

Western blot

Sample

Human Cell lysate - whole cell (human hepatocytes)

Gel Running Conditions

Reduced Denaturing

Loading amount

20 µg

Treatment

10 ng/ml TNF-a+100 nM Smac mimetic+20 µM z-VAD 6 h

Specification

human hepatocytes

Blocking step

Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Jun 21 2018

Application

Western blot

Sample

Human Cell lysate - whole cell (Prosate cancer cell line)

Gel Running Conditions

Reduced Denaturing (10)

Loading amount

20 µg

Specification

Prosate cancer cell line

Blocking step

BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 23°C

Abcam user community

Verified customer

Submitted May 02 2018

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

Abcam user community

Verified customer

Submitted Jan 31 2018

1-10 of 13 Abreviews or Q&A

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Key features and details

Related conjugates and formulations

Overview

Product name

Description

Host species

Specificity

Tested applications

Species reactivity

Immunogen

Positive control

General notes

Properties

Form

Storage instructions

Storage buffer

Purity

Clonality

Clone number

Isotype

Research areas

Associated products

Alternative Versions

Compatible Secondaries

Immunizing Peptide (Blocking)

Isotype control

Recombinant Protein

Related Products

Applications

The Abpromise guarantee

Target

Sequence similarities

Domain

Database links

Alternative names

Images

Protocols

Datasheets and documents

SDS download

Datasheet download

Certificate of Compliance

References (131)

Customer reviews and Q&As

Western blot abreview for Anti-MLKL (phospho S358) antibody [EPR9514]

Immunocytochemistry/ Immunofluorescence abreview for Anti-MLKL (phospho S358) antibody [EPR9514]

Immunohistochemistry (Frozen sections) abreview for Anti-MLKL (phospho S358) antibody [EPR9514]

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

Western blot abreview for Anti-MLKL (phospho S358) antibody [EPR9514]

Western blot abreview for Anti-MLKL (phospho S358) antibody [EPR9514]

Western blot abreview for Anti-MLKL (phospho S358) antibody [EPR9514]

Western blot abreview for Anti-MLKL (phospho S358) antibody [EPR9514]

Western blot abreview for Anti-MLKL (phospho S358) antibody [EPR9514]

Immunocytochemistry abreview for Anti-MLKL (phospho S358) antibody [EPR9514]