Lane 1: Wild type HAP1 whole cell lysate (20 µg)              
Lane 2: UCHL1 (KO) knockout  HAP1 whole cell lysate (20 µg)
Lane 3: SH-SY5Y whole cell lysate (20 µg)
Lane 4: Hek293 whole cell lysate (20 µg)              

Lanes 1 - 4: Merged signal (red and green). Green - ab20559 observed at 24 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab20559 detected the expected band for PGP9.5 in wild type cells and the band was not seen in PGP9.5/UCHL1 knockout cells. Wild-type and UCHL1 knockout samples were subjected to SDS-PAGE.  Ab20559 and ab181602 (Rabbit anti GAPDH loading control) were incubated overnight at 4°C at 1 ug/ml and 1/10000 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 1/10000 dilution for 1 hour at room temperature before imaging.

Overlay histogram showing SH-SH5Y cells stained with ab20559 (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 (ab20559, 0.1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150113) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 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. This antibody gave a positive signal in SH-SY5Y cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

All lanes : Anti-PGP9.5 antibody [31A3] (ab20559) at 5 µg/ml

Lane 1 : Human brain tissue lysate - total protein (ab29466)
Lane 2 : Human spinal cord tissue lysate - total protein (ab29188)
Lane 3 : Brain (Rat) Tissue Lysate
Lane 4 : Brain (Mouse) Tissue Lysate
Lane 5 : SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 25 kDa
Additional bands at: 25 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 4 minutes