Anti-CD276 antibody [EPNCIR122] - Low endotoxin, Azide free

• Flow Cyt

Lab

Flow Cytometry - Anti-CD276 antibody [EPNCIR122] - Low endotoxin, Azide free (AB209895)

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).

• Flow Cyt

Unknown

Flow Cytometry - Anti-CD276 antibody [EPNCIR122] - Low endotoxin, Azide free (AB209895)

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.

• Flow Cyt

Unknown

Flow Cytometry - Anti-CD276 antibody [EPNCIR122] - Low endotoxin, Azide free (AB209895)

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).

• IP

Lab

Immunoprecipitation - Anti-CD276 antibody [EPNCIR122] - Low endotoxin, Azide free (AB209895)

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).

All lanes:

Immunoprecipitation - Anti-CD276 antibody [EPNCIR122] (<a href='/en-us/products/primary-antibodies/cd276-antibody-epncir122-ab134161'>ab134161</a>)

Predicted band size: 57 kDa

false

Exposure time: 3s

• IHC-Fr

Collaborator

Immunohistochemistry (Frozen sections) - Anti-CD276 antibody [EPNCIR122] - Low endotoxin, Azide free (AB209895)

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).

Image from Seaman S et al. Cancer Cell, 31, 501-515. Fig2.D doi:10.1016/j.ccell.2017.03.005. with permission from Elsevier

• WB

Unknown

Western blot - Anti-CD276 antibody [EPNCIR122] - Low endotoxin, Azide free (AB209895)

Blocking buffer and concentration : 5% NFDM/TBST

Diluting buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-CD276 antibody [EPNCIR122] - Low endotoxin, Azide free (ab209895)

All lanes:

LNCaP (human prostat carcinoma) 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>)

Predicted band size: 57 kDa

Observed band size: 110 kDa

false

Exposure time: 3min