Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-HADHA antibody [EPR17940] (ab203114)

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Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR17940] to HADHA
  • Suitable for: ICC/IF, IP, Flow Cyt, IHC-P, WB
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Overview

  • Product name

    Anti-HADHA antibody [EPR17940]
    See all HADHA primary antibodies

  • Description

    Rabbit monoclonal [EPR17940] to HADHA

  • Host species

    Rabbit

  • Tested applications

    Suitable for: ICC/IF, IP, Flow Cyt, IHC-P, WBmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Human

  • Immunogen

    Synthetic peptide within Human HADHA aa 600-700. The exact sequence is proprietary.
    Database link: P40939

  • Positive control

    • WB: HeLa, HEK-293, HepG2, Jurkat, C6 and RAW 264.7 cell lysates; Human fetal liver and fetal kidney lysates; Mouse heart and kidney lysates; Rat heart and kidney lysates. IHC-P: Human cervix carcinoma and tonsil tissues. ICC/IF: Jurkat, HeLa cells, HAP1 wildtype and HAP1-HADHA knockout cells Flow Cyt: HeLa cells. IP: HeLa whole cell lysate.

  • General notes

     

     

    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.

Properties

Applications

Our Abpromise guarantee covers the use of ab203114 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 Use a concentration of 1 µg/ml.
IP 1/40.
Flow Cyt 1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

 
IHC-P 1/100. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
WB 1/1000. Detects a band of approximately 74 kDa (predicted molecular weight: 83 kDa).

Target

  • Function

    Bifunctional subunit.

  • Pathway

    Lipid metabolism; fatty acid beta-oxidation.

  • Involvement in disease

    Defects in HADHA are a cause of trifunctional protein deficiency (TFP deficiency) [MIM:609015]. The clinical manifestations are very variable and include hypoglycemia, cardiomyopathy and sudden death. Phenotypes with mainly hepatic and neuromyopathic involvement can also be distinguished. Biochemically, TFP deficiency is defined by the loss of all enzyme activities of the TFP complex.
    Defects in HADHA are the cause of long-chain 3-hydroxyl-CoA dehydrogenase deficiency (LCHAD deficiency) [MIM:609016]. The clinical features are very similar to TFP deficiency. Biochemically, LCHAD deficiency is characterized by reduced long-chain 3-hydroxyl-CoA dehydrogenase activity, while the other enzyme activities of the TFP complex are normal or only slightly reduced.
    Defects in HADHA are a cause of maternal acute fatty liver of pregnancy (AFLP) [MIM:609016]. AFLP is a severe maternal illness occurring during pregnancies with affected fetuses. This disease is associated with LCHAD deficiency and characterized by sudden unexplained infant death or hypoglycemia and abnormal liver enzymes (Reye-like syndrome).

  • Sequence similarities

    In the N-terminal section; belongs to the enoyl-CoA hydratase/isomerase family.
    In the central section; belongs to the 3-hydroxyacyl-CoA dehydrogenase family.

  • Cellular localization

    Mitochondrion.
  • Information by UniProt

  • Database links

    see all

  • Alternative names

    • 3 ketoacyl Coenzyme A (CoA) thiolase alpha subunit antibody
    • 3 oxoacyl CoA thiolase antibody
    • 78 kDa gastrin binding protein antibody
    • 78 kDa gastrin-binding protein antibody
    • ECHA antibody
    • ECHA_HUMAN antibody
    • GBP antibody
    • HADH antibody
    • HADHA antibody
    • Hydroxyacyl Coenzyme A dehydrogenase/3 ketoacyl Coenzyme A thiolase/enoyl Coenzyme A hydratase (trifunctional protein) alpha subunit antibody
    • LCEH antibody
    • LCHAD antibody
    • Long chain 3-hydroxyacyl-CoA dehydrogenase antibody
    • Mitochondrial long chain 2 enoyl Coenzyme A (CoA) hydratase alpha subunit antibody
    • Mitochondrial long chain L 3 hydroxyacyl Coenzyme A dehydrogenase alpha subunit antibody
    • Mitochondrial trifunctional enzyme alpha subunit antibody
    • Mitochondrial trifunctional protein alpha subunit antibody
    • MTPA antibody
    • Thiolase/enoyl Coenzyme A hydratase (trifunctional protein) alpha subunit antibody
    • TP ALPHA antibody
    • TP-alpha antibody
    • Trifunctional enzyme subunit alpha mitochondrial precursor antibody
    see all

Images

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

    Lane 1 : Wild-type HEK-293T cell lysate
    Lane 2 : HADHA knockout HEK-293T cell lysate
    Lane 3 : HepG2 cell lysate
    Lane 4 : SH-SY5Y cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 83 kDa
    Observed band size: 82 kDa
    why is the actual band size different from the predicted?



    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.

     

  • Immunocytochemistry/ Immunofluorescence - Anti-HADHA antibody [EPR17940] (ab203114)
    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).

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

    Lane 1 : Wild-type HAP1 cell lysate
    Lane 2 : HADHA knockout HAP1 cell lysate
    Lane 3 : HEK293 cell lysate
    Lane 4 : HepG2 cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 83 kDa



    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.

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

    Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) cell lysate
    Lane 2 : HEK-293 (Human epithelial cells from embryonic kidney) cell lysate
    Lane 3 : HepG2 (Human liver hepatocellular carcinoma) cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

    Predicted band size: 83 kDa
    Observed band size: 74 kDa why is the actual band size different from the predicted?


    Exposure time: 3 minutes


    Blocking/Dilution buffer: 5% NFDM/TBST.

  • Western blot - Anti-HADHA antibody [EPR17940] (ab203114)
    Western blot - Anti-HADHA antibody [EPR17940] (ab203114)
    Anti-HADHA antibody [EPR17940] (ab203114) at 1/1000 dilution + Jurkat (Human T cell leukemia cells from peripheral blood) cell lysate at 20 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

    Predicted band size: 83 kDa
    Observed band size: 74 kDa why is the actual band size different from the predicted?


    Exposure time: 3 minutes


    Blocking/Dilution buffer: 5% NFDM/TBST.

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

    Lane 1 : Human fetal liver lysate
    Lane 2 : Human fetal kidney lysate

    Lysates/proteins at 10 µg per lane.

    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 why is the actual band size different from the predicted?


    Exposure time: 30 seconds


    Blocking/Dilution buffer: 5% NFDM/TBST.

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

    Lane 1 : Mouse heart lysate
    Lane 2 : Mouse kidney lysate
    Lane 3 : Rat heart lysate
    Lane 4 : Rat kidney lysate
    Lane 5 : C6 (Rat glial tumor cells) cell lysate
    Lane 6 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

    Predicted band size: 83 kDa
    Observed band size: 74 kDa why is the actual band size different from the predicted?


    Exposure time: 15 seconds


    Blocking/Dilution buffer: 5% NFDM/TBST.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HADHA antibody [EPR17940] (ab203114)
    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.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HADHA antibody [EPR17940] (ab203114)
    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.

  • Immunocytochemistry/ Immunofluorescence - Anti-HADHA antibody [EPR17940] (ab203114)
    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.

  • Immunocytochemistry/ Immunofluorescence - Anti-HADHA antibody [EPR17940] (ab203114)
    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 Cytometry - Anti-HADHA antibody [EPR17940] (ab203114)
    Flow Cytometry - Anti-HADHA antibody [EPR17940] (ab203114)

    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.

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

References

Publishing research using ab203114? Please let us know so that we can cite the reference in this datasheet.

ab203114 has been referenced in 4 publications.

  • Fisher-Wellman KH  et al. Respiratory Phenomics across Multiple Models of Protein Hyperacylation in Cardiac Mitochondria Reveals a Marginal Impact on Bioenergetics. Cell Rep 26:1557-1572.e8 (2019). PubMed: 30726738
  • Che L  et al. Valine increases milk fat synthesis in mammary gland of gilts through stimulating AKT/MTOR/SREBP1 pathway‡. Biol Reprod N/A:N/A (2019). PubMed: 30985894
  • Chen J  et al. The Potential of the FSP1cre-Pparb/d-/- Mouse Model for Studying Juvenile NAFLD. Int J Mol Sci 20:N/A (2019). PubMed: 31618976
  • Liang K  et al. Cryo-EM structure of human mitochondrial trifunctional protein. Proc Natl Acad Sci U S A 115:7039-7044 (2018). PubMed: 29915090

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