Anti-FACL4 antibody [EPR17587-42]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-FACL4 antibody [EPR17587-42] (AB205199)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling FACL4 with ab205199 at 1/120 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/500 dilution was used as the secondary antibody.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-FACL4 antibody [EPR17587-42] (AB205199)

Immunofluorescent analysis of 4% paraformaldehyde-fixed 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling FACL4 with ab205199 at 1/50 dilution followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HeLa cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).
The negative controls are as follows;
-ve control 1 : ab205199 at 1/50 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.
-ve control 2 : ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-FACL4 antibody [EPR17587-42] (AB205199)

Immunofluorescent analysis of 4% paraformaldehyde-fixed 0.1% Triton X-100 permeabilized HepG2 (Human liver hepatocellular carcinoma) cells labeling FACL4 with ab205199 at 1/50 dilution followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HepG2 cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).
The negative controls are as follows;
-ve control 1 : ab205199 at 1/50 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.
-ve control 2 : ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FACL4 antibody [EPR17587-42] (AB205199)

Immunohistochemical analysis of paraffin-embedded Human liver carcinoma tissue labeling FACL4 with ab205199 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Cytoplasmic staining on Human hepatocellular carcinoma tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

Unknown

Immunoprecipitation - Anti-FACL4 antibody [EPR17587-42] (AB205199)

FACL4 was immunoprecipitated from 1mg of Mouse brain whole cell lysate with ab205199 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab205199 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 : Mouse brain whole cell lysate 10 μg (Input). Lane 2 : ab205199 IP in Mouse brain whole cell lysate. Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab205199 in Mouse brain whole cell lysate
Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 8 seconds

All lanes:

Immunoprecipitation - Anti-FACL4 antibody [EPR17587-42] (ab205199)

Predicted band size: 79 kDa

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• WB

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Western blot - Anti-FACL4 antibody [EPR17587-42] (AB205199)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-FACL4 antibody [EPR17587-42] (ab205199) at 1/5000 dilution

Lane 1:

HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

HepG2 (Human liver hepatocellular carcinoma) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 44 kDa,79 kDa

Observed band size: 24 kDa,44 kDa,72 kDa,79 kDa

false

Exposure time: 30s

• WB

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Western blot - Anti-FACL4 antibody [EPR17587-42] (AB205199)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-FACL4 antibody [EPR17587-42] (ab205199) at 1/1000 dilution

Lane 1:

Human fetal brain lysate at 10 µg

Lane 2:

Human fetal heart lysate at 10 µg

Lane 3:

Human fetal kidney lysate at 10 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 79 kDa

Observed band size: 79 kDa

false

Exposure time: 1min

• WB

Supplier Data

Western blot - Anti-FACL4 antibody [EPR17587-42] (AB205199)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-FACL4 antibody [EPR17587-42] (ab205199) at 1/5000 dilution

All lanes:

293 (Human epithelial cells from embryonic kidney) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 79 kDa

Observed band size: 79 kDa

false

Exposure time: 3min

• WB

CiteAb

Western blot - Anti-FACL4 antibody [EPR17587-42] (AB205199)

FACL4 western blot using anti-FACL4 antibody [EPR17587-42] ab205199. Publication image and figure legend from Jiang, Y., Zhao, X., et al., 2020, Nat Commun, PubMed 32312987.

ab205199 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab205199 please see the product overview.

In vitro NIR-II photothermal ferrotherapy.a Confocal laser scanning microscopy (CLSM) images of different types of cancer cells and normal cells after incubation with HSN ([pTBCB] = 50 µg mL−1) or PBS for 24 h. ROS was indicated by green fluorescence from DCF-DA staining. Nuclei were stained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) and indicated by blue fluorescence. 4T1 : murine mammary carcinoma cell line; MCF-7 : human breast adenocarcinoma cell line; HepG2 : human hepatocellular carcinoma cell line; NIH/3T3 : murine fibroblast cell line; NDF : normal human dermal fibroblast cell line. b Western immunoblot analysis of expression levels of ferroptosis-related proteins (ACSL4, FPN-1, GPX4) in a panel of cancer cell lines. MDA-MB-231 (231) : human breast adenocarcinoma cell line; PC12 : rat pheochromocytoma cell line; HeLa : human cervical adenocarcinoma cell line; SKOV3 : human ovarian adenocarcinoma cell line. Source data were provided in Source Data File. c CLSM images of 4T1 cells after incubation with PBS, HSN, HSN with apoptosis inhibitor DEVD (100 µM), HSN with ferroptosis inhibitor deferoxamine (DFO) (100 µM) for 24 h, respectively. [pTBCB] = 50 µg mL−1. Expression of Cas-3 was indicated by immunofluorescence staining (green fluorescence), and lipid peroxidation was stained with a red-fluorescent probe BODIPY 665/676. Cell viabilities (d) and relative GSH levels (e) of 4T1 cells after incubation with HSN0 or HSN at various concentrations for 24 h with or without 1064 nm photoirradiation (1 W cm−2, 6 min). [pTBCB] = 50 µg mL−1. f Western immunoblots analysis of expression levels of ferroptosis and apoptosis related proteins in 4T1 cells in d and e. Plus and minus symbol indicated with and without photoirradiation, respectively. Source data were provided in Source Data File. g Proposed molecular mechanisms of HSN-mediated NIR-II photothermal ferrotherapy. GSSG glutathione disulfide, AA arachidonic acid, AA-CoA arachidonyl-CoA, LH phospholipid. Error bars indicated standard deviations of three independent measurements.

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