Anti-CD276 antibody [EPNCIR122] - BSA and Azide free

• Flow Cyt

Lab

Flow Cytometry - Anti-CD276 antibody [EPNCIR122] - BSA and Azide free (AB256585)

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).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-CD276 antibody [EPNCIR122] - BSA and Azide free (AB256585)

This data was developed using the same antibody clone in a different buffer formulation (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).

• Flow Cyt

Unknown

Flow Cytometry - Anti-CD276 antibody [EPNCIR122] - BSA and Azide free (AB256585)

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.

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] - BSA and Azide free (AB256585)

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/TBSTPerformed 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] - BSA and Azide free (AB256585)

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

Supplier Data

Western blot - Anti-CD276 antibody [EPNCIR122] - BSA and Azide free (AB256585)

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].

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

All lanes:

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