Name: Anti-MLKL (phospho S345) antibody [EPR9515(2)]
Brand: RabMAb
SKU: ab196436
Rating: 4 (17 reviews)

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Protein

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Primary - Rabbit Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436)
ICC/IF, WB, IP, Dot blot

Secondary - Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
IHC-P, WB, ELISA, IP

Protein - Recombinant Human MLKL protein (ab153623)
WB, ELISA

View more associated products

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.
General notes
This antibody was developed through collaboration with the lab of Xiaodong Wang at the National Institute of Biological Sciences, Beijing.
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

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)
Recombinant Protein
- Recombinant Human MLKL protein (ab153623)
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 annonymous Abreview

Paraformaldehyde-fixed, triton X-100 permeabilized Rat Cell (primary hippocampal neurons treated with TNF-a/ZVA) labeling MLKL (phospho S345) (Green) using ab196436 at 1/100 dilution in ICC/IF analysis. A Invitrogen A11034 Goat polyclonal Alexa Fluor® 488 was used as a secondary antibody.
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.
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
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:

Patel S et al. RIP1 inhibition blocks inflammatory diseases but not tumor growth or metastases. Cell Death Differ 27:161-175 (2020). Read more (PubMed: 31101885) »
Kadigamuwa C et al. Role of Retinoic Acid Receptor-? in DNA Damage-Induced Necroptosis. iScience 17:74-86 (2019). Read more (PubMed: 31255985) »

See all 78 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 17 Abreviews or Q&A

Application

IHC - Wholemount

Sample

Mouse Tissue (Brain isolated capillaries)

Specification

Brain isolated capillaries

Abcam user community

Verified customer

Submitted Sep 19 2019

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

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

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

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

1-10 of 17 Abreviews or Q&A

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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

Isotype control

Recombinant Protein

Related Products

Applications

Target

Sequence similarities

Domain

Database links

Alternative names

Images

Protocols

Datasheets and documents

Certificate of Compliance

References

Customer reviews and Q&As

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

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)]