Anti-Syntenin antibody [EPR8102]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Syntenin antibody [EPR8102] (AB133267)

ab133267 was shown to react with SDCBP in wild-type HAP1 cells in immunocytochemistry with loss of signal observed in a SDCBP siRNA knockdown cell line. Wild-type and siRNA knockdown cells were mixed and pelleted at a 1 : 1 ratio on coverslips. The cells were fixed with 4% paraformaldehyde (15 min) then permeabilized with 0.1% Triton X-100 (10min) and then blocked with 1x PBS, 0.01% Triton X-100, 5% BSA, 5% NGS. The cells were then incubated with ab133267 at 1/500 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat anti-rabbit secondary antibody to (Alexa Fluor® 555) at 0.5 μg/ml. Acquisition of the green (wild-type), red (antibody staining) and far-red (siRNA knockdown) channels was performed. Representative grayscale images of the red channel are shown. Wild-type and siRNA knockdown cells are outlined with yellow and magenta dashed line, respectively. Schematic representation of the mosaic strategy used is shown on the bottom-right panel. Image was acquired with a Zeiss(LSM-880).

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Syntenin antibody [EPR8102] (AB133267)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Syntenin antibody [EPR8102] (AB133267)

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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Syntenin antibody [EPR8102] (AB133267)

Intracellular Flow Cytometry analysis ofHeLa (Human epithelial cell line from cervix adenocarcinoma)labeling Syntenin with purifiedab133267 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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Syntenin antibody [EPR8102] (AB133267)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Syntenin antibody [EPR8102] (AB133267)

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Syntenin antibody [EPR8102] (AB133267)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Syntenin antibody [EPR8102] (AB133267)

Immunohistochemical analysis of formalin fixed paraffin embedded human liver carcinoma labelling Syntenin with ab133267 at a concentration of 0.01µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab133267 Anti-Syntenin antibody [EPR8102] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• IP

Lab

Immunoprecipitation - Anti-Syntenin antibody [EPR8102] (AB133267)

Immunoprecipitation of SDCBP in HAP1 culture medium. Culture medium was prepared and immunoprecipitation was performed using 2 μg of ab133267 pre-coupled to Protein A beads. Samples were then washed and processed for western blot.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Immunoprecipitation - Anti-Syntenin antibody [EPR8102] (ab133267) at 2 µg

All lanes:

HAP1 culture medium

Observed band size: 31 kDa

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• IP

Lab

Immunoprecipitation - Anti-Syntenin antibody [EPR8102] (AB133267)

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.

All lanes:

Immunoprecipitation - Anti-Syntenin antibody [EPR8102] (ab133267)

Predicted band size: 32 kDa

Observed band size: 32 kDa

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• IP

Lab

Immunoprecipitation - Anti-Syntenin antibody [EPR8102] (AB133267)

Immunoprecipitation of SDCBP in HAP1 cells. Lysates were prepared and immunoprecipitation was performed using 2 μg of ab133267 pre-coupled to Protein A beads. Samples were then washed and processed for western blot.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Immunoprecipitation - Anti-Syntenin antibody [EPR8102] (ab133267) at 2 µg

All lanes:

HAP1 cells lysates

Observed band size: 31 kDa

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• WB

Lab

Western blot - Anti-Syntenin antibody [EPR8102] (AB133267)

Blocking and dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Syntenin antibody [EPR8102] (ab133267) at 1/2000 dilution

All lanes:

A549 (Human lung carcinoma cell line) whole cell lysate at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 32 kDa

Observed band size: 32 kDa

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• WB

Lab

Western blot - Anti-Syntenin antibody [EPR8102] (AB133267)

Blocking and dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Syntenin antibody [EPR8102] (ab133267) at 1/2000 dilution

Lane 1:

Human placenta lysate at 15 µg

Lane 2:

Human fetal brain lysate at 15 µg

Secondary

All lanes:

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

Predicted band size: 32 kDa,46 kDa,86 kDa

Observed band size: 32 kDa

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• WB

Lab

Western blot - Anti-Syntenin antibody [EPR8102] (AB133267)

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.

All lanes:

Western blot - Anti-Syntenin antibody [EPR8102] (ab133267)

Predicted band size: 32 kDa

false

• WB

Lab

Western blot - Anti-Syntenin antibody [EPR8102] (AB133267)

ab133267 was shown to react with SDCBP in wild-type HAP1 cells in Western blot with loss of signal observed in a SDCBP siRNA knockdown cell line. Cell lysates from wild-type HAP1 transfected with either scrambled siRNA or SDCBP siRNA were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab133267 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-Syntenin antibody [EPR8102] (ab133267) at 1/1000 dilution

Lane 1:

Wild-type HAP1 transfected with scrambled siRNA control lysate at 30 µg

Lane 2:

HAP1 transfected with siRNA specifically targeting SDCBP cell lysate at 30 µg

Observed band size: 31 kDa

false

• WB

Lab

Western blot - Anti-Syntenin antibody [EPR8102] (AB133267)

ab133267 was shown to react with SDCBP in wild-type HAP1 culture medium in Western blot with loss of signal observed in a SDCBP siRNA knockdown culture media. Cell culture medium from wild-type HAP1 transfected with either scrambled siRNA or SDCBP siRNA was subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab133267 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-Syntenin antibody [EPR8102] (ab133267) at 1/1000 dilution

Lane 1:

Wild-type HAP1 transfected with scrambled siRNA control culture medium at 30 µg

Lane 2:

HAP1 transfected with siRNA specifically targeting SDCBP culture medium at 30 µg

Observed band size: 31 kDa

false

• WB

Lab

Western blot - Anti-Syntenin antibody [EPR8102] (AB133267)

Blocking and dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Syntenin antibody [EPR8102] (ab133267) at 1/10000 dilution

All lanes:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 110 kDa,119 kDa,14 kDa,15 kDa,25 kDa,32 kDa,43 kDa

Observed band size: 11 kDa,15 kDa,25 kDa,32 kDa

false

• WB

Unknown

Western blot - Anti-Syntenin antibody [EPR8102] (AB133267)

All lanes:

Western blot - Anti-Syntenin antibody [EPR8102] (ab133267) at 1/1000 dilution

Lane 1:

Human fetal brain lysate at 10 µg

Lane 2:

293T cell lysate at 10 µg

Lane 3:

Human fetal heart lysate at 10 µg

Lane 4:

HeLa cell lysate at 10 µg

Secondary

All lanes:

Goat anti-rabbit HRP conjugated antibody at 1/2000 dilution

Predicted band size: 32 kDa

Observed band size: 32 kDa

false

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-Syntenin antibody [EPR8102] (AB133267)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.