Anti-HADHA antibody [EPR17939]

• Flow Cyt (Intra)

Lab

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)

• ICC/IF

Lab

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.

• ICC/IF

Lab

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.

• IP

Supplier Data

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.

All lanes:

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

Predicted band size: 83 kDa

false

• WB

Supplier Data

Western blot - Anti-HADHA antibody [EPR17939] (AB200652)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-HADHA antibody [EPR17939] (ab200652) at 1/10000 dilution

All lanes:

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

Secondary

All lanes:

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

Predicted band size: 62 kDa,66 kDa,83 kDa

Observed band size: 62 kDa,74 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-HADHA antibody [EPR17939] (AB200652)

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.

All lanes:

Western blot - Anti-HADHA antibody [EPR17939] (ab200652) 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

Lab

Western blot - Anti-HADHA antibody [EPR17939] (AB200652)

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.

All lanes:

Western blot - Anti-HADHA antibody [EPR17939] (ab200652) 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

false

• WB

Supplier Data

Western blot - Anti-HADHA antibody [EPR17939] (AB200652)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

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

Lane 1:

Human fetal brain lysate at 10 µg

Lane 2:

Human fetal kidney lysate at 10 µg

Lane 3:

Human fetal liver lysate at 10 µg

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

false

Exposure time: 1s

• WB

Supplier Data

Western blot - Anti-HADHA antibody [EPR17939] (AB200652)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

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

Lane 1:

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

Lane 2:

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

Lane 3:

HEK293 (Human embryonic kidney) whole cell lysate at 20 µg

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

false

Exposure time: 1s

• WB

Supplier Data

Western blot - Anti-HADHA antibody [EPR17939] (AB200652)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

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

Lane 1:

Mouse kidney lysate at 10 µg

Lane 2:

Rat heart lysate at 10 µg

Lane 3:

Rat kidney lysate at 10 µg

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

false

Exposure time: 3min