All lanes : Anti-Lactate Dehydrogenase B/LDH-B antibody [EP1565Y] (ab53292) at 1/5000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : LDHB knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 37 kDa
Observed band size: 37 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab53292 observed at 37 kDa. Red - Anti-Vinculin antibody [VIN-54] observed at 124 kDa.

 ab53292 was shown to react with Lactate Dehydrogenase B/LDH-B in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab255403 (knockout cell lysate ab263761) was used. Wild-type HEK-293T and LDHB knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab53292 and Anti-Vinculin antibody [VIN-54] overnight at 4°C at a 1 in 5000 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.

ICC/IF image of ab53292 stained HepG2 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab53292, neat) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

All lanes : Anti-Lactate Dehydrogenase B/LDH-B antibody [EP1565Y] (ab53292) at 1/50000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : LDHB (Lactate Dehydrogenase B) knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 37 kDa

Lanes 1 - 3: Merged signal (red and green). Green - ab53292 observed at 37 kDa. Red - loading control, ab130007, observed at 130 kDa.

ab53292 was shown to specifically react with Lactate Dehydrogenase B in wild-type HAP1 cells as signal was lost in LDHB (Lactate Dehydrogenase B) knockout cells. Wild-type and LDHB (Lactate Dehydrogenase B) knockout samples were subjected to SDS-PAGE. Ab53292 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/50000 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.

Human heart muscle labeled with ab53292 at 1/100 - 1/250 dilution

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Anti-Lactate Dehydrogenase B/LDH-B antibody [EP1565Y] (ab53292) + HeLa cell lysate at 10 µg

Secondary
Goat anti-rabbit HRP

Predicted band size: 37 kDa
Observed band size: 37 kDa

Overlay histogram showing HeLa cells stained with ab53292 (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 (ab53292, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.