All lanes : Anti-Alkaline Phosphatase, Tissue Non-Specific antibody [2F4] (ab126820) at 1/500 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : ALPL knockout HAP1 whole cell lysate
Lane 3 : Saos2 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 57, 78.9 kDa

Lanes 1 - 3: Merged signal (red and green). Green - ab126820 observed at 80 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab126820 was shown to specifically react with ALPL in wild-type HAP1 cells as signal was lost in ALPL knockout cells. Wild-type and ALPL knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab126820 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 1/500 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 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.

Anti-Alkaline Phosphatase, Tissue Non-Specific antibody [2F4] (ab126820) at 1/500 dilution + Recombinant Human protein ALPL (AA: 18-502)

Predicted band size: 57, 78.9 kDa

ICC/IF image of ab126820 stained HepG2 cells. The cells were 4% formaldehyde fixed (10 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 ab126820 at 1/100 dilution overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- mouse (ab96879) 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. This antibody also gave a positive result in methanol fixed (100%, 5min) hepG2 cells used at 1/100 dilution.

All lanes : Anti-Alkaline Phosphatase, Tissue Non-Specific antibody [2F4] (ab126820) at 1/500 dilution

Lane 1 : HeLa cell lysate
Lane 2 : NTERA-2 cell lysate

Predicted band size: 57, 78.9 kDa

Example of standard curves obtained using ab126820. Control antigen 100ng (black), antigen at 10ng (purple), antigen at 50ng (blue), antigen at 100ng (red)

Flow cytometric analysis of MCF-7 cells using ab126820 at 1/200 (green) and negative control (red).

ab126820, at 1/200, staining Alkaline Phosphatase, Tissue Non-Specific in paraffin embedded Human ovarian cancer tissues by Immunohistochemistry with DAB staining.