All lanes : Anti-PDHA1 antibody [EPR11099] (ab155096) at 1/1000 dilution

Lane 1 : Wild-type HeLa whole cell lysate
Lane 2 : PDHA1 knockout HeLa whole cell lysate
Lane 3 : HEK-293 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 43 kDa
Observed band size: 43 kDa

Lanes 1 - 3: Merged signal (red and green). Green - ab155096 observed at 43 kDa. Red - loading control, ab130007, observed at 130 kDa.

ab155096 was shown to recognize PDHA1 in wild-type HeLa cells as signal was lost at the expected MW in PDHA1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and PDHA1 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab155096 and ab130007 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-PDHA1 antibody [EPR11099] (ab155096) at 1/1000 dilution

Lane 1 : HepG2 cell lysate
Lane 2 : 293T cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : Jurkat cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 43 kDa

Overlay histogram showing HeLa cells stained with ab155096 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab155096, 1/10000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

Immunohistochemical analysis of paraffin-embedded Human thyroid carcinoma tissue labeling PDHA1 with ab155096 at 1/50 dilution.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunofluorescent analysis of HeLa cells labeling PDHA1 with ab155096 at 1/250 dilution.