Anti-PGP9.5 抗体 [EPR4118] (ab108986)

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 - ab108986 observed at 24 kDa. Red - loading control, ab8245, observed at 37 kDa.

Ab108986 was shown to specifically react with UCHL1 (KO) in wild-type cells as signal was lost in UCHL1 (KO) knockout HAP1 cells. Wild-type and UCHL1 (KO) knockout samples were subjected to SDS-PAGE.  Ab108986 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Neuro-2a (Mouse neuroblastoma cell line) cells labeling PGP9.5 with ab108986 at 1/500 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on Neuro-2a cell line. The nuclear counter stain is DAPI (blue).

The negative control is PBS only.

All lanes : Anti-PGP9.5 antibody [EPR4118] (ab108986) at 1/1000 dilution

Lane 1 : Fetal brain cell lysate
Lane 2 : Y79 cell lysate
Lane 3 : U87-MG cell lysate
Lane 4 : SH-SY5Y cell lysate
Lane 5 : 293T cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit IgG at 1/2000 dilution

Predicted band size: 24 kDa
Observed band size: 25 kDa (why is the actual band size different from the predicted?)

Overlay histogram showing SH-SY5Y cells stained with ab108986 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab108986, 1/10000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/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 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

ab108986 staining PGP9.5 in human colon tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 2% BSA for 10 minutes at 21°C; antigen retrieval was by heat mediation in citric acid. Samples were incubated with the primary antibody (1/500 in TBS/BSA/azide) for 16 hours at 21°C. A Biotin-conjugated goat anti-rabbit IgG polyclonal (1/300) was used as the secondary antibody.

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Immunohistochemical analysis of mouse colon tissue sections labeling PGP9.5 with ab108986 at a dulution of 1/1500. Sections were fixed with Formaldehyde. A Biotin conjugated Goat Anti-Rabbit IgG at 1/300 was used as the secondary antibody. Antigen retrieval was heat mediated using citric acid. 

All nerve cell/fibre components of enteric plexuses are demonstrated very well.

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Immunohistochemical analysis of rat Jejunum tissue sections labeling PGP9.5 with ab108986 at a dulution of 1/1000. Sections were fixed with Formaldehyde. A Biotin conjugated Goat Anti-Rabbit IgG at 1/300 was used as the secondary antibody. Antigen retrieval was heat mediated using citric acid. 

All nerve components of enteric plexuses appear to be very well demonstrated, particularly the fine fibres of the lamina propria and the muscularis mucosa.

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Immunohistochemical staining of PGP9.5 in paraffin embedded Human glioma tissue, using ab108986 at a 1/250 dilution.