Name: Anti-MLKL (phospho S345) antibody [EPR9515(2)]
Brand: RabMAb
SKU: ab196436
Rating: 4 (16 reviews)

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: ICC/IF, WB, IP, Dot blotmore details
Species reactivity

Reacts with: Mouse
Immunogen

Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) within Mouse MLKL aa 300-400 (phospho S345). The exact sequence is proprietary.
Database link: Q9D2Y4
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. ICC/IF: Mouse cardiomyocytes.
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

EPR9515(2)
Isotype

IgG
Research areas

Alternative Versions
- Anti-MLKL (phospho S345) antibody [EPR9515(2)] (HRP) (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)
Related Products

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
ICC/IF		1/100.
WB		1/1000. Detects a band of approximately 54 kDa (predicted molecular weight: 54 kDa).
IP		1/150.
Dot blot		1/1000.

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

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.
Immunocytochemistry/ Immunofluorescence - Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436)This image is courtesy of an anonymous Abreview

Ab196436 staining MLKL in mouse cardiomyocytes by Immunocytochemistry. Samples were fixed with paraformaldehyde, permeabilized with 0.03% Triton x-100 and blocked with 1% BSA for 1 hour at room temperature. Samples were incubated with primary antibody at 1/100 dilution for 12 hours. An Alexa Fluor^® anti- rabbit was used as a secondary antibody at 1/200 dilution.
See Abreview
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)

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 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.

Western blot protocols
Immunoprecipitation protocols

This product has been referenced in:

Yuan Z et al. A new perspective of triptolide-associated hepatotoxicity: Liver hypersensitivity upon LPS stimulation. Toxicology 414:45-56 (2019). Read more (PubMed: 30633930) »
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 40 Publications for this product

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

Customer reviews and Q&As

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1-10 of 16 Abreviews or Q&A

Application

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

Sample

Rat Tissue sections (brain)

Antigen retrieval step

Heat mediated - Buffer/Enzyme Used: Sodium citrate

Permeabilization

Specification

brain

Blocking step

BSA as blocking agent for 15 minute(s) · Concentration: 1% · Temperature: RT°C

Fixative

Paraformaldehyde

Abcam user community

Verified customer

Submitted Aug 01 2018

Abcam has not validated the combination of species/application used in this Abreview.

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

Mrs. Francoise Geffroy

Verified customer

Submitted Mar 05 2018

Application

Immunocytochemistry

Sample

Mouse Cultured Cells (Cardiomyocytes)

Permeabilization

Yes - Triton x-100, 0.03%

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

Western blot

Sample

Chinese hamster Cell lysate - whole cell (Chinese hamster ovary (CHO) cells)

Gel Running Conditions

Reduced Denaturing (4-12% Bolt Bis-Tris Plus gel (MES buffer))

Loading amount

50000 cells

Specification

Chinese hamster ovary (CHO) cells

Blocking step

Li-Cor Odyssey Blocking Buffer (TBS) as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Dec 31 2016

Abcam has not validated the combination of species/application used in this Abreview.

Application

Immunocytochemistry/ Immunofluorescence

Sample

Rat Cell (primary hippocampal neurons treated with TNF-a/ZVA)

Permeabilization

Yes - triton X-100

Specification

primary hippocampal neurons treated with TNF-a/ZVA

Blocking step

Gelatin from cold water fish skin as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Fixative

Paraformaldehyde

Abcam user community

Verified customer

Submitted Dec 20 2016

Application

Western blot

Sample

Rabbit Cell lysate - whole cell (Rabbit Reticulocyte)

Gel Running Conditions

Reduced Denaturing (4-12% Bolt Bis-Tris Plus gel (MES buffer))

Loading amount

0.5 µg

Specification

Rabbit Reticulocyte

Blocking step

Li-Cor Odyssey Blocking Buffer (TBS) as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Dec 12 2016

Application

Western blot

Sample

Rat Tissue lysate - whole (Brain)

Gel Running Conditions

Reduced Denaturing (10%)

Loading amount

40 µg

Specification

Brain

Blocking step

Milk as blocking agent for 40 minute(s) · Concentration: 5% · Temperature: 22°C

Abcam user community

Verified customer

Submitted Oct 27 2016

Application

Western blot

Sample

Rat Tissue lysate - whole (thymus)

Gel Running Conditions

Reduced Denaturing (4-15% BioRad TGX gel)

Loading amount

15 µg

Treatment

saline (CT) vs. methamphetamine (MT) 25mg/kg i.p., 6hr survival

Specification

thymus

Blocking step

Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Abcam user community

Verified customer

Submitted Sep 05 2016

Application

Western blot

Sample

Human Cell lysate - whole cell (A549 (human lung epithelium cells))

Gel Running Conditions

Reduced Denaturing (4-12% Bolt Bis-Tris Plus gel)

Loading amount

50000 cells

Treatment

TNFalpha (25ng/mL) and zVAD (50uM) for 5 hours

Specification

A549 (human lung epithelium cells)

Blocking step

Li-Cor Odyssey Blocking Buffer (TBS) as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Aug 10 2016

Application

Western blot

Sample

African Green Monkey Cell lysate - whole cell (VERO E6)

Gel Running Conditions

Reduced Denaturing (4-12% Bolt Bis-Tris Plus gel)

Loading amount

75000 cells

Treatment

TNFalpha (25ng/mL) and zVAD (50uM) for 5 hours

Specification

VERO E6

Blocking step

Li-Cor Odyssey Blocking Buffer (TBS) as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Aug 09 2016

1-10 of 16 Abreviews or Q&A

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

Overview

Properties

Associated products

Applications

Target

Images

Protocols

Datasheets and documents

References

This product has been referenced in:

Customer reviews and Q&As

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

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

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

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

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

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

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

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

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

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