Anti-CD276 antibody [EPNCIR122] - Low endotoxin, Azide free (ab209895) + LNCaP (human prostat carcinoma) whole cell lysate at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051)

Predicted band size: 57 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking buffer and concentration: 5% NFDM/TBST

Diluting buffer and concentration: 5% NFDM/TBST

Flow cytometry overlay histogram showing wild-type HEK293 (green line) and CD276 knockout HEK293 (ab266658) cells stained with ab209895 (red line). The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab209895) (1x10⁶ in 100μl at 0.2 μg/ml) for 30 min at 4°C.

The secondary antibody Goat anti-rabbit IgG H&L (Alexa Fluor^® 488, pre-adsorbed) (ab150081) was used at 1/2000 for 30 min at 4°C.

Isotype control antibody was Rabbit IgG (monoclonal) (ab172730) used at the same concentration and conditions as the primary antibody (wild-type HEK293 - black line CD276 HEK293 knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

Immunohistochemistry of mouse MC38 colon liver metastasis. Staining CD276 with ab134161 (green). Normal tissue (N)/tumor tissue (T) margins are indicated by a white dash.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134161).

CD276 was immunoprecipitated from 10 ug of HEK 293 (human embryonic kidney epithelial cell) whole cell lysate with ab134161 at 1/40 diltuion. Western blot was permformed from the immunoprecipitate using ab 134161 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/500 dilution.

Lane 1: HEK 293 whole cell lysate 10 ug (Input).

Lane 2: ab134161 IP in HEK 293 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab134161 in HEK 293 whole cell lysate.

Blocking/dilution buffer: 5% NFDM/TBST

Performed using purified antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134161).

Overlay histogram showing THP-1 (human monocytic leukemia monocyte) cells stained with ab134161 (red line). The cells were fixed with 4% paraformaldehyde and then permeabilized with 90% methanol. The cells were incubated with the antibody (ab134161) at 1/80 dilution. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution. Isotype control antibody (black line) was rabbit monoclonal IgG (ab172730) used under the same conditions. Unlabelled sample (blue line) was also used as a control.

Performed using purified antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134161).

Overlay histogram showing THP1 cells stained with ab134161 (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% human serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab134161, 1/1000 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 data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134161).