All lanes : Anti-GLO1 antibody [6F10] (ab81461) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : GLO1 knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : HepG2 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 21 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab81461 observed at 21 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab81461 was shown to specifically react with in wild-type HAP1 cells as signal was lost in GLO1 knockout cells. Wild-type and GLO1 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab81461 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rat IgG H&L (IRDye® 800CW) preabsorbed and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-GLO1 antibody [6F10] (ab81461)

Lane 1 : COS-1 (simian) whole cell extract
Lane 2 : L929 (mouse) whole cell extract
Lane 3 : HeLa (human) whole cell extract

Predicted band size: 21 kDa
Observed band size: 27-29 kDa why is the actual band size different from the predicted?



Mouse GLO1 shows a single band of 27 kDa while human and simian ones show 29 kDa.

Immunofluorescent staining of HeLa cells with ab81461.

Overlay histogram showing HeLa cells stained with ab81461 (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 (ab81461, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rat IgG (H+L) (ab98386) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rat IgG2b [RTK4530] (ab18541, 1µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line). Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.