Anti-GLO1 antibody [6F10]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-GLO1 antibody [6F10] (AB81461)

Immunofluorescent staining of HeLa cells with ab81461 at a 1/500 dilution. Cells fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. As the second antibody, goat anti-rat IgG antibody conjugated with FITC was used at 1/5,000 dilution.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-GLO1 antibody [6F10] (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 22°C. The secondary antibody used was DyLight^® 488 goat anti-rat IgG (H+L) (ab98386) at 1/500 dilution for 30 min at 22°C. 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.

• WB

Supplier Data

Western blot - Anti-GLO1 antibody [6F10] (AB81461)

Mouse (COS-1) GLO1 shows a single band of 27 kDa while human (HeLa) and simian (L929) ones show 29 kDa.

All lanes:

Western blot - Anti-GLO1 antibody [6F10] (ab81461) at 1/1000 dilution

Lane 1:

COS-1 (simian) whole cell extract

Lane 2:

L929 (mouse) whole cell extract

Lane 3:

HeLa (human) whole cell extract

Secondary

All lanes:

Goat anti-rat IgG antibody conjugated with HRP at 1/10000 dilution

Predicted band size: 21 kDa

Observed band size: 27-29 kDa

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• WB

Lab

Western blot - Anti-GLO1 antibody [6F10] (AB81461)

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:

Western blot - Anti-GLO1 antibody [6F10] (ab81461) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

GLO1 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Lane 4:

HepG2 whole cell lysate at 20 µg

Predicted band size: 21 kDa

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