Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-HADHA antibody [EPR17939] (ab200652)

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Overview

  • Product name

    Anti-HADHA antibody [EPR17939]
    See all HADHA primary antibodies

  • Description

    Rabbit monoclonal [EPR17939] to HADHA

  • Host species

    Rabbit

  • Tested applications

    Suitable for: IP, ICC/IF, WB, Flow Cytmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Human

  • Immunogen

    Synthetic peptide within Human HADHA aa 250-350. The exact sequence is proprietary.
    Database link: P40939

  • Positive control

    • WB: HeLa, Jurkat, HEK293 and HepG2 whole cell lysates; Human fetal brain, fetal kidney and fetal liver lysates; Mouse kidney, rat heart and rat kidney lysates. ICC/IF: Jurkat and HeLa cells. IP: HEK293 whole cell lysate. Flow: Jurkat (human acute T cell leukemia)

  • 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 ab200652 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
IP 1/30.
ICC/IF 1/250.
WB 1/1000. Detects a band of approximately 74 kDa (predicted molecular weight: 83 kDa).
Flow Cyt Use a concentration of 1 µg/ml.

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 [EPR17939] (ab200652)
    Western blot - Anti-HADHA antibody [EPR17939] (ab200652)
    All lanes : Anti-HADHA antibody [EPR17939] (ab200652) at 1/1000 dilution

    Lane 1 : Wild-type HEK-293T cell lysate
    Lane 2 : H2AFY 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 - ab200652 observed at 82 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

    ab200652 was shown to react with HADHA in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout sample ab257464 was used. Wild-type HEK-293T and H2AFY Knockout HEK-293T cell lysates were subjected to SDS-PAGE. ab200652 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 Dilution and a 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 [EPR17939] (ab200652)
    Immunocytochemistry/ Immunofluorescence - Anti-HADHA antibody [EPR17939] (ab200652)

    Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling HADHA with ab200652 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green).

    Cytoplasm staining on HeLa cell line is observed.

    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 1/500 dilution (red).
    The negative controls are as follows:
    -ve control 1: ab200652 at 1/250 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 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 1/500 dilution.

     

  • Western blot - Anti-HADHA antibody [EPR17939] (ab200652)
    Western blot - Anti-HADHA antibody [EPR17939] (ab200652)
    All lanes : Anti-HADHA antibody [EPR17939] (ab200652) 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 - ab200652 observed at 82 kDa. Red - loading control, ab8245, observed at 37 kDa.
    ab200652 was shown to specifically react with HADHA when HADHA knockout samples were used. Wild-type and HADHA knockout samples were subjected to SDS-PAGE. ab200652 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 1/10 000 dilution for 1 h at room temperature before imaging.

  • Western blot - Anti-HADHA antibody [EPR17939] (ab200652)
    Western blot - Anti-HADHA antibody [EPR17939] (ab200652)
    All lanes : Anti-HADHA antibody [EPR17939] (ab200652) at 1/1000 dilution

    Lane 1 : Mouse kidney lysate
    Lane 2 : Rat heart lysate
    Lane 3 : Rat kidney lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 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.

  • Flow Cytometry - Anti-HADHA antibody [EPR17939] (ab200652)
    Flow Cytometry - Anti-HADHA antibody [EPR17939] (ab200652)

    ab200652 staining HADHA in Jurkat (human acute T cell leukemia) cells by flow cytometry. Cells were fixed with 4% paraformaldehyde and the sample was incubated with the primary antibody at a dilution of 1/2200. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/2000 was used as the secondary antibody.

    Isoytype control: Rabbit monoclonal IgG (Black)

    Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)

  • Western blot - Anti-HADHA antibody [EPR17939] (ab200652)
    Western blot - Anti-HADHA antibody [EPR17939] (ab200652)
    All lanes : Anti-HADHA antibody [EPR17939] (ab200652) at 1/1000 dilution

    Lane 1 : Human fetal brain lysate
    Lane 2 : Human fetal kidney lysate
    Lane 3 : Human fetal liver lysate

    Lysates/proteins at 10 µg per lane.

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

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


    Exposure time: 1 second


    Blocking/Dilution buffer: 5% NFDM/TBST.

  • Immunocytochemistry/ Immunofluorescence - Anti-HADHA antibody [EPR17939] (ab200652)
    Immunocytochemistry/ Immunofluorescence - Anti-HADHA antibody [EPR17939] (ab200652)

    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 ab200652 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green).

    Cytoplasm staining on Jurkat cell line is observed.

    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 1/500 dilution (red).
    The negative controls are as follows:
    -ve control 1: ab200652 at 1/250 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 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 1/500 dilution.

     

  • Western blot - Anti-HADHA antibody [EPR17939] (ab200652)
    Western blot - Anti-HADHA antibody [EPR17939] (ab200652)
    Anti-HADHA antibody [EPR17939] (ab200652) at 1/10000 dilution + HepG2 (Human liver hepatocellular carcinoma) whole cell lysate at 20 µg

    Secondary
    Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 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 [EPR17939] (ab200652)
    Western blot - Anti-HADHA antibody [EPR17939] (ab200652)
    All lanes : Anti-HADHA antibody [EPR17939] (ab200652) at 1/1000 dilution

    Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
    Lane 2 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate
    Lane 3 : HEK293 (Human embryonic kidney) whole cell lysate

    Lysates/proteins at 20 µg per lane.

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

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


    Exposure time: 1 second


    Blocking/Dilution buffer: 5% NFDM/TBST.

  • Immunoprecipitation - Anti-HADHA antibody [EPR17939] (ab200652)
    Immunoprecipitation - Anti-HADHA antibody [EPR17939] (ab200652)

    HADHA was immunoprecipitated from 1mg of HEK293 (Human embryonic kidney) whole cell lysate with ab200652 at 1/30 dilution.

    Western blot was performed from the immunoprecipitate using ab200652 at 1/2000 dilution.

    Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

    Lane 1: HEK293 whole cell lysate 10 µg (Input).

    Lane 2: ab200652 IP in HEK293 whole cell lysate.

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab200652 in HEK293 whole cell lysate.

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

References

Publishing research using ab200652? Please let us know so that we can cite the reference in this datasheet.

ab200652 has not yet been referenced specifically in any publications.

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