Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: Syntenin knockout HAP1 whole cell lysate (20 µg)
Lane 3: A549 whole cell lysate (20 µg)
Lane 4: HeLa whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab133267 observed at 32 kDa. Red - loading control, ab130007, observed at 130 kDa.

ab133267 was shown to recognize Syntenin in wild-type HAP1 cells as signal was lost at the expected MW in Syntenin knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and Syntenin knockout samples were subjected to SDS-PAGE. ab133267 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human hepatocellular carcinoma tissue labeling Syntenin with purified ab133267 at 1/50 dilution. Heat mediated antigen retrieval was performed using EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin

Anti-Syntenin antibody [EPR8102] (ab133267) at 1/2000 dilution (purified) + A549 (Human lung carcinoma cell line) whole cell lysate at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 32 kDa
Observed band size: 32 kDa

Blocking and dilution buffer: 5% NFDM/TBST

Flow Cytometry analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) labeling Syntenin with purified ab133267 at 1/50 (red). Cells were fixed with 4% paraformaldehyde. A goat anti rabbit IgG (Alexa Fluor® 488) 1/2000 was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

ab133267 (purified) at 1/40 immunoprecipitating Syntenin in HeLa whole cell lysate.

Lane 1 (input): HeLa whole cell lysate (10µg)

Lane 2 (+): ab133267 + HeLa whole cell lysate.

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab133267 in HeLa whole cell lysate.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

 

 

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human cerebral cortex tissue labeling Syntenin with purified ab133267 at 1/50 dilution. Heat mediated antigen retrieval was performed using EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

All lanes : Anti-Syntenin antibody [EPR8102] (ab133267) at 1/2000 dilution (purified)

Lane 1 : Human placenta lysate
Lane 2 : Human fetal brain lysate

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution

Predicted band size: 32 kDa
Observed band size: 32 kDa

Blocking and dilution buffer: 5% NFDM/TBST

Anti-Syntenin antibody [EPR8102] (ab133267) at 1/10000 dilution (purified) + HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution

Predicted band size: 32 kDa
Observed band size: 32 kDa

Blocking and dilution buffer: 5% NFDM/TBST

ab133267 staining Syntenin in HeLa (human cervix adenocarcinoma) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/500. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at a dilution of 1/1000. DAPI was used as a nuclear counterstain.

Negative control 1: PBS only.

Overlay histogram showing SHSY-5Y cells stained with unpurified ab133267 (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 (ab133267, 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 SHSY-5Y cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

All lanes : Anti-Syntenin antibody [EPR8102] (ab133267) at 1/1000 dilution (unpurified)

Lane 1 : Human fetal brain lysate
Lane 2 : 293T cell lysate
Lane 3 : Human fetal heart lysate
Lane 4 : HeLa cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat anti-rabbit HRP conjugated antibody at 1/2000 dilution

Predicted band size: 32 kDa
Observed band size: 32 kDa

Immunohistochemical analysis of Syntenin in paraffin embedded Human brain tissue, using unpurified ab133267 at a 1/50 dilution.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Equilibrium disassociation constant (K_D)
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