Antibodies, Proteins, Kits and Reagents for Life Science

324 988 ₸ Product size

100 µl 324 988 ₸

Order now and get it on Tuesday March 09, 2021

Anti-HADHA antibody [EPR17939] (ab200652)

Key features and details

Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
Rabbit monoclonal [EPR17939] to HADHA
Suitable for: IP, ICC/IF, WB, Flow Cyt
Knockout validated
Reacts with: Mouse, Rat, Human

Product name

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

Rabbit monoclonal [EPR17939] to HADHA
Host species

Rabbit

Tested Applications & Species

Application	Species
Flow Cyt	Human
ICC/IF	Human
IP	Human
WB	Mouse Rat Human

See all applications and species data

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.
Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.

Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.

We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.

In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.

We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.

Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.

Please check that this product meets your needs before purchasing. If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, as well as customer reviews and Q&As.

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

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 - 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)

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)

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