Anti-PGP9.5 antibody [346CT2.5.1] (ab86808)

IHC-P image of PGP9.5 staining on cat bladder sections using ab86808 (1:3000). The sections were subjected to heat mediated antigen retrieval using citric acid. The sections were then blocked using 1% BSA at 21°C for 10 min. ab86808 was incubated at 21°C for 1 hours. The secondary antibody was Goat polyclonal to anti-mouse (rat pre-absorbed) conjugated to biotin (1:200).

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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)

Lanes 1 - 3: Merged signal (red and green). Green - ab86808 observed at 24 kDa. Red - loading control, ab181602, observed at 37 kDa.

Ab86808 was shown to specifically react with PGP9.5/UCHL1 in wild-type cells as signal was lost in PGP9/UCHL1 knockout HAP1 cells. Wild-type and UCHL1 (KO) knockout samples were subjected to SDS-PAGE.  Ab86808 and ab181602 (Rabbit anti GAPDH loading control) were incubated overnight at 4°C at 1 ug/ml and 1/2000 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 ab86808 (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 (ab86808, 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.

IHC-P image of PGP9.5 staining using ab86808(1:2500) on dog small intestine sections. The sections were subjected to heat mediated antigen retrieval using citric acid. The sections were then blocked with 1% BSA for 10 min at 21°C. the sections were then incubated with primary antibody ab86808 at 1:2500 dlution for 2 hours at 21°C. The secondary used was Goat polyclonal to anti mouse IgG conjugated to biotin (1:200)

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All lanes : Anti-PGP9.5 antibody [346CT2.5.1] (ab86808) at 1 µg/ml

Lane 1 : Human brain tissue lysate - total protein (ab29466)
Lane 2 : Brain (Rat) Tissue Lysate
Lane 3 : Rat Cortex Tissue Lysate
Lane 4 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate
Lane 5 : Y79 (Human retinoblastoma cell line) Whole Cell Lysate
Lane 6 : U-87 MG (Human glioblastoma astrocytoma) Whole Cell Lysate
Lane 7 : 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 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 25 kDa
Observed band size: 25 kDa


Exposure time: 1 minute

IHC-P image of PGP9.5 staining on rat pancreas using ab86808 (1:1000). The rat pancreatic sections were subjected to heat mediated antigen retrieval using citric acid. The sections were then blocked using 1% BSA at 21°C for 10 min. ab86808 was incubated at 21°C for 1 hours. The secondary antibody was Goat polyclonal to anti-mouse (rat pre-absorbed) conjugated to biotin (1:200).

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