Anti-PGP9.5 antibody [31A3]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-PGP9.5 antibody [31A3] (AB20559)

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.

• WB

Lab

Western blot - Anti-PGP9.5 antibody [31A3] (AB20559)

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.

All lanes:

Western blot - Anti-PGP9.5 antibody [31A3] (ab20559)

Predicted band size: 24 kDa

false

• WB

Lab

Western blot - Anti-PGP9.5 antibody [31A3] (AB20559)

All lanes:

Western blot - Anti-PGP9.5 antibody [31A3] (ab20559) at 5 µg/mL

Lane 1:

Human brain tissue lysate - total protein (<a href='/en-us/products/unavailable/human-brain-tissue-lysate-total-protein-ab29466'>ab29466</a>) at 20 µg

Lane 2:

Human spinal cord tissue lysate - total protein (<a href='/en-us/products/unavailable/human-spinal-cord-tissue-lysate-total-protein-ab29188'>ab29188</a>) at 20 µg

Lane 3:

Brain (Rat) Tissue Lysate at 20 µg

Lane 4:

Brain (Mouse) Tissue Lysate at 20 µg

Lane 5:

SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Mouse IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-hrp-preadsorbed-ab97040'>ab97040</a>) at 1/50000 dilution

Predicted band size: 24 kDa

Observed band size: 25 kDa

true

Exposure time: 4min