Anti-CD276 antibody [EPNCIR122]

• WB

Lab

Western blot - Anti-CD276 antibody [EPNCIR122] (AB134161)

Blocking/dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-CD276 antibody [EPNCIR122] (ab134161) at 0.264 µg/mL

Lane 1:

CHO-K1 cell lysates, transfected with Human CD276 at 20 µg

Lane 2:

CHO-K1 cell lysates, transfected with Mouse CD276. at 20 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: 57 kDa

false

Exposure time: 3min

• Flow Cyt

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Flow Cytometry - Anti-CD276 antibody [EPNCIR122] (AB134161)

Flow cytometry overlay histogram showing wild-type HEK293 (green line) and CD276 knockout HEK293 (ab266658) cells stained with ab134161 (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 (ab134161) (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

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Flow Cytometry - Anti-CD276 antibody [EPNCIR122] (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 >5000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

• Flow Cyt

Lab

Flow Cytometry - Anti-CD276 antibody [EPNCIR122] (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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CD276 antibody [EPNCIR122] (AB134161)

ab134161 staining CD276 in wild-type HEK293 cells (top panel) and CD276 knockout HEK293 cells (ab266658) (bottom panel). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab134161 at 1μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

• IP

Lab

Immunoprecipitation - Anti-CD276 antibody [EPNCIR122] (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.

All lanes:

Immunoprecipitation - Anti-CD276 antibody [EPNCIR122] (ab134161)

Predicted band size: 57 kDa

false

Exposure time: 3s

• IHC-Fr

Collaborator

Immunohistochemistry (Frozen sections) - Anti-CD276 antibody [EPNCIR122] (AB134161)

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.

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

Lab

Western blot - Anti-CD276 antibody [EPNCIR122] (AB134161)

Blocking/dilution buffer : 5% NFDM/TBST

Performed using purified antibody.

All lanes:

Western blot - Anti-CD276 antibody [EPNCIR122] (ab134161) at 1/1000 dilution

All lanes:

LNCap (human prostate carcinoma epithelial cell) 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: 112 kDa,388 kDa,47 kDa,55 kDa,57 kDa,72 kDa,83 kDa,97 kDa

Observed band size: 100 kDa,110 kDa,140 kDa,388 kDa,50-70 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-CD276 antibody [EPNCIR122] (AB134161)

We recommend not to boil the samples after lysis to get desired WB results.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

CD276 (B7-H3) has a molecular weight of about 90–100 kDa, depending on the glycosylation levels. [PMID : 22473715].

All lanes:

Western blot - Anti-CD276 antibody [EPNCIR122] (ab134161) at 1/1000 dilution

Lane 1:

HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate boiled. at 20 µg

Lane 2:

HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate unboiled. at 20 µg

Lane 3:

LNCaP (Human prostate carcinoma epithelial cell) whole cell lysate boiled. at 20 µg

Lane 4:

LNCaP (Human prostate carcinoma epithelial cell) whole cell lysate unboiled. at 20 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: 57 kDa

Observed band size: 90 kDa

false

• WB

Supplier Data

Western blot - Anti-CD276 antibody [EPNCIR122] (AB134161)

Blocking and dilution buffer : 5% NFDM/TBST.

We recommend not to boil the samples after lysis to get desired WB results.

Mouse CD276 (B7-H3) has a molecular weight about 45–66 kDa, depending on the glycosylation levels [PMID : 34366684].

All lanes:

Western blot - Anti-CD276 antibody [EPNCIR122] (ab134161) at 1/1000 dilution

Lane 1:

LLC (Mouse Lewis lung carcinoma cell) whole cell lysate (boiled) at 20 µg

Lane 2:

LLC (Mouse Lewis lung carcinoma cell) whole cell lysate (unboiled) at 20 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: 57 kDa

Observed band size: 45-66 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-CD276 antibody [EPNCIR122] (AB134161)

Blocking/dilution buffer : 5% NFDM/TBST

Performed using purified antibody.

All lanes:

Western blot - Anti-CD276 antibody [EPNCIR122] (ab134161) at 1/200 dilution

All lanes:

CHO (hamster ovary epithelial cell) whole cell lysates transfected with human CD276 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 1/10000 dilution

Predicted band size: 57 kDa

Observed band size: 110 kDa

false

Exposure time: 3min