Recombinant Anti-HADHA antibody [EPR17939] KO Tested (ab200652)

Product name

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

Rabbit monoclonal [EPR17939] to HADHA
Host species

Rabbit
Tested applications

Suitable for: Flow Cyt (Intra), IP, ICC/IF, WBmore details
Species reactivity

Reacts with: Mouse, Rat, Human
Immunogen

Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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.

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

pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Concentration information loading...
Purity

Protein A purified
Clonality

Monoclonal
Clone number

EPR17939
Isotype

IgG
Research areas

Alternative Versions
- Anti-HADHA antibody [EPR17939] - BSA and Azide free (ab242411)
Compatible Secondaries
- Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
- Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
Isotype control
- Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
KO cell lines
- Human HADHA knockout HEK-293T cell line (ab266274)
KO cell lysates
- Human HADHA knockout HEK-293T cell lysate (ab257464)
Recombinant Protein
- Recombinant Human HADHA protein (ab158631)

The Abpromise guarantee

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

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

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.
Target information above from: UniProt accession P40939 The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010) .

Information by UniProt
Database links
- Entrez Gene: 3030 Human
- Entrez Gene: 97212 Mouse
- Entrez Gene: 170670 Rat
- Omim: 600890 Human
- SwissProt: P40939 Human
- SwissProt: Q8BMS1 Mouse
- SwissProt: Q64428 Rat
- Unigene: 516032 Human
- Unigene: 200497 Mouse
- Unigene: 3340 Rat
- Unigene: 34751 Rat
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

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 : 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 - ab200652 observed at 82 kDa. Red - loading control ab8245 observed at 37 kDa.

ab200652 Anti-HADHA antibody [EPR17939] 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. ab200652 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 [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)

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)

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 (Intracellular) - Anti-HADHA antibody [EPR17939] (ab200652)

ab200652 staining HADHA in Jurkat (human acute T cell leukemia) cellsby intracellular 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)

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)

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)

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)

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)

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.
Anti-HADHA antibody [EPR17939] (ab200652)

Flow cytometry protocols
Immunoprecipitation protocols
Immunocytochemistry & immunofluorescence protocols
Western blot protocols

Click here to view the general protocols

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Key features and details

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Overview

Product name

Description

Host species

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

KO cell lines

KO cell lysates

Recombinant Protein

Applications

The Abpromise guarantee

Target

Function

Pathway

Involvement in disease

Sequence similarities

Cellular localization

Database links

Alternative names

Images

Protocols

Datasheets and documents

Datasheet download

Certificate of Compliance

References (0)

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