Anti-HADHA antibody [EPR17940]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-HADHA antibody [EPR17940] (AB203114)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling HADHA with ab203114 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on Jurkat cell line.

The nuclear counter stain is DAPI (blue). COX IV is detected with ab33985 (anti-COX IV Mitochondrial Marker 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 : ab203114 at 1/250 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.
-ve control 2 : ab33985 (anti-COX IV Mitochondrial Marker mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HADHA antibody [EPR17940] (AB203114)

Immunohistochemical analysis of paraffin-embedded Human cervix carcinoma tissue labeling HADHA with ab203114 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasmic staining on Human cervix 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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HADHA antibody [EPR17940] (AB203114)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling HADHA with ab203114 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasmic staining on Human tonsil 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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-HADHA antibody [EPR17940] (AB203114)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling HADHA with ab203114 at 1/250 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). COX IV is detected with ab33985 (anti-COX IV Mitochondrial Marker 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 : ab203114 at 1/250 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.
-ve control 2 : ab33985 (anti-COX IV Mitochondrial Marker mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-HADHA antibody [EPR17940] (AB203114)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling HADHA with ab203114 at 1/100 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

Lab

Immunocytochemistry/ Immunofluorescence - Anti-HADHA antibody [EPR17940] (AB203114)

ab203114 staining HADHA in wild-type HAP1 cells (top panel) and HADHA knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol for 5 minutes, permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1 hour. The cells were then incubated with ab203114 at 1μg/ml concentration dilution and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1 hour with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) secondary antibody at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• IP

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Immunoprecipitation - Anti-HADHA antibody [EPR17940] (AB203114)

HADHA was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate with ab203114 at 1/400 dilution. Western blot was performed from the immunoprecipitate using ab203114 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 : HeLa whole cell lysate 10ug (Input). Lane 2 : ab203114 IP in HeLa whole cell lysate. Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab203114 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 3 seconds.

All lanes:

Immunoprecipitation - Anti-HADHA antibody [EPR17940] (ab203114)

Predicted band size: 83 kDa

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• WB

Lab

Western blot - Anti-HADHA antibody [EPR17940] (AB203114)

Lanes 1 - 4 : Merged signal (red and green). Green - ab203114 observed at 82 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab203114 was shown to specifically react with HADHA when HADHA knockout samples were used. Wild-type and HADHA knockout samples were subjected to SDS-PAGE. ab203114 and ab8245 (loading control to GAPDH) were diluted at 1/1000 and 1/10 000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-HADHA antibody [EPR17940] (ab203114) at 1/1000 dilution

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

HADHA knockout HAP1 cell lysate at 20 µg

Lane 3:

HEK293 cell lysate at 20 µg

Lane 4:

HepG2 cell lysate at 20 µg

Predicted band size: 83 kDa

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• WB

Supplier Data

Western blot - Anti-HADHA antibody [EPR17940] (AB203114)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-HADHA antibody [EPR17940] (ab203114) at 1/1000 dilution

Lane 1:

Human fetal liver lysate at 10 µg

Lane 2:

Human fetal kidney lysate at 10 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/50000 dilution

Predicted band size: 83 kDa

Observed band size: 74 kDa

false

Exposure time: 30s

• WB

Supplier Data

Western blot - Anti-HADHA antibody [EPR17940] (AB203114)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-HADHA antibody [EPR17940] (ab203114) at 1/10000 dilution

Lane 1:

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

Lane 2:

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

Lane 3:

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

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 83 kDa

Observed band size: 74 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-HADHA antibody [EPR17940] (AB203114)

Lanes 1-4 : Merged signal (red and green). Green - ab203114 observed at 82 kDa. Red - loading control ab8245 observed at 37 kDa.

ab203114 Anti-HADHA antibody [EPR17940] was shown to specifically react with HADHA in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266274 (knockout cell lysate ab257464) was used. Wild-type and HADHA knockout samples were subjected to SDS-PAGE. ab203114 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-HADHA antibody [EPR17940] (ab203114) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

HADHA knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human HADHA knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-hadha-knockout-hek-293t-cell-line-ab266274'>ab266274</a>)

Lane 3:

HepG2 cell lysate at 20 µg

Lane 4:

SH-SY5Y cell lysate at 20 µg

Predicted band size: 83 kDa

Observed band size: 82 kDa

false

• WB

Supplier Data

Western blot - Anti-HADHA antibody [EPR17940] (AB203114)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-HADHA antibody [EPR17940] (ab203114) at 1/1000 dilution

All lanes:

Jurkat (Human T cell leukemia cells from peripheral blood) cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 83 kDa

Observed band size: 74 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-HADHA antibody [EPR17940] (AB203114)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-HADHA antibody [EPR17940] (ab203114) at 1/2000 dilution

Lane 1:

Mouse heart lysate at 10 µg

Lane 2:

Mouse kidney lysate at 10 µg

Lane 3:

Rat heart lysate at 10 µg

Lane 4:

Rat kidney lysate at 10 µg

Lane 5:

C6 (Rat glial tumor cells) cell lysate at 10 µg

Lane 6:

RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 83 kDa

Observed band size: 74 kDa

false

Exposure time: 15s

• WB

CiteAb

Western blot - Anti-HADHA antibody [EPR17940] (AB203114)

HADHA western blot using anti-HADHA antibody [EPR17940] ab203114. Publication image and figure legend from Chen, J., Zhuang, Y., et al., 2019, Int J Mol Sci, PubMed 31618976.

ab203114 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 ab203114 please see the product overview.

Expression levels of proteins in fatty acid β-oxidation, fatty acid synthesis, and triglyceride synthesis pathways. Immunoblot analysis of indicated proteins in the liver of P2 Pparb/dfl/fl and FSP1cre-Pparb/d-/- mice. Representative blots from 7 mice (biological replicates) for each genotype and the results of the 6-7 mice (biological replicates) are shown in bar graphs. Two-tailed Mann-Whitney test with values shown as mean ± s.e.m. * p < 0.05, ** p < 0.01; FSP1cre-Pparb/d-/- vs. Pparb/dfl/fl controls. (a) HADHA. β-actin was used as loading and transfer control. (b) Phospho-ACLY and ACLY. β-tubulin was used as loading and transfer control. (c) Phospho-ACC and ACC. β-tubulin was used as loading and transfer control. (d) GPD2. β-actin was used as loading and transfer control. The full size original western blots are shown in Figure SM2.

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