Anti-CD276 抗体 [RM2041] (ab315907)

Negative control: Raji(PMID: 21344157).

In Western blot, ab315907 was shown to bind specifically to CD276.

Target of interest was observed at 90 KD in wild-type HEK293T cell lysates with no signal observed at this size in CD276 knockout cell line (lane 2, knockout cell line ab266658/ knockout cell lysate ab257097).

Lysates were freshly made, non-boiled and used immediately to minimize protein degradation.

In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade  (ab176842) staining at 1/100000 dilution.

Lysates were freshly made, non-boiled and used immediately to minimize protein degradation.

Low expression: skeletal muscle(PMID:36756390, PMID:35476450)

This blot was developed using a higher sensitivity ECL substrate.

Samples are non-boiled as boiling may cause protein aggregation.

The identity of the lower MW bands in lane 1 and 2 are unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control  (ab181602) staining at 1/200000 dilution.

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling CD276 with ab315907 at 1/200 (2.505 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human tonsil. The section was incubated with ab315907 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling CD276 with ab315907 at 1/200 (2.505 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human breast carcinoma. The section was incubated with ab315907 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Human cardiac muscle tissue labeling CD276 with ab315907 at 1/200 (2.505 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control: no staining on human cardiac muscle. The section was incubated with ab315907 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Human skeletal muscle tissue labeling CD276 with ab315907 at 1/200 (2.505 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression: no staining on human skeletal muscle. The section was incubated with ab315907 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized CD276 KO HEK293T (CD276 knockout human embryonic kidney epithelial cell) cells labelling CD276 with ab315907 at 1/100 (5.01 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing membranous staining in parental HEK293T cells, and no staining in CD276 KO HEK293T cells. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

Flow cytometric analysis of CD276 KO HEK293T (human CD276 knockout embryonic kidney epithelial cell, Left) / Parental HEK293T(Right) cells labelling CD276 with ab315907 at 1/5000 dilution (0.01 ug)/Red compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells.

CD276 was immunoprecipitated from 0.35 mg 293T (human embryonic kidney epithelial cell) whole cell lysate with ab315907 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab315907 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: 293T (human embryonic kidney epithelial cell) whole cell lysate
Lane 2: ab315907 IP in 293T (human embryonic kidney epithelial cell) whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab315907 in 293T whole cell lysate

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

Exposure time: 84 seconds.