Name: Anti-MLKL (phospho S358) antibody [EPR9514]
Brand: RabMAb
SKU: ab187091
Rating: 4 (6 reviews)

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 (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.
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.

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

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)
- Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
Immunizing Peptide (Blocking)
- Human MLKL (phospho S358) peptide (ab206929)
Isotype control
- Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
Related Products
- Prestained Protein Ladder - Broad molecular weight (10-245 kDa) (ab116028)

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).
Dot blot		1/1000.
IHC-P		1/250 - 1/500. 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
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
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)

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).

Western blot protocols
Immunohistochemistry protocols
Protocol Booklet

Datasheet

This product has been referenced in:

Mishra SK et al. Upregulation of human glycolipid transfer protein (GLTP) induces necroptosis in colon carcinoma cells. Biochim Biophys Acta Mol Cell Biol Lipids 1864:158-167 (2019). Read more (PubMed: 30472325) »
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 50 Publications for this product

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

Customer reviews and Q&As

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1-6 of 6 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.

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

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

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

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

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

Specification

Liver

Blocking step

Ventana blocking agent as blocking agent for 4 minute(s) · Concentration: 100% · Temperature: 36°C

Fixative

Formaldehyde

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

Overview

Properties

Associated products

Applications

Target

Images

Protocols

Datasheets and documents

References

This product has been referenced in:

Customer reviews and Q&As

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]

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

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

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