Anti-ErbB2 / HER2 antibody [CAL27]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-ErbB2 / HER2 antibody [CAL27] (AB237715)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilised SK-BR-3 (human mammary gland adenocarcinoma cell line) cells labeling ERbB 2 with ab237715 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous staining in SK-BR-3 cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ErbB2 / HER2 antibody [CAL27] (AB237715)

IHC image of ErbB2 / HER2 staining in a section of formalin-fixed paraffin-embedded MCF7 cell pellet performed on a Leica Biosystems BOND® RX instrument using the standard protocol. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab237715, 1/200 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ErbB2 / HER2 antibody [CAL27] (AB237715)

Negative control image : IHC image of ErbB2 / HER2 staining in a section of formalin-fixed paraffin-embedded MCF7 ErbB2 / HER2 KO cell pellet performed on a Leica Biosystems BOND® RX instrument using the standard protocol. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab237715, 1/200 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ErbB2 / HER2 antibody [CAL27] (AB237715)

IHC image of ErbB2 / HER2 staining in a section of formalin-fixed paraffin-embedded normal human breast cancer* performed on a Leica Biosystems BOND® RX instrument using the standard protocol. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab237715, 1/200 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ErbB2 / HER2 antibody [CAL27] (AB237715)

Formalin-fixed, paraffin-embedded human breast carcinoma tissue stained for ErbB2 / HER2 using ab237715 at 0.3 μg/ml in immunohistochemical analysis.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ErbB2 / HER2 antibody [CAL27] (AB237715)

Immunohistochemical analysis of human breast carcinoma tissue labeling ErbB2 / HER2 with ab237715 at 1/2000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on the human breast carcinoma is observed. Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

The section was incubated with ab237715 for 10 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-ErbB2 / HER2 antibody [CAL27] (AB237715)

Intracellular flow cytometric analysis of4% paraformaldehyde-fixed, 90% methonao-permeabilized SH-BR-3 (human mammary gland adenocarcinoma cell line) cells labeling ErbB2 / HER2 with ab237715 at 1/500 dilution (red) compared with Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue).

Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077), at 1/2000 dilution was used as the secondary antibody.

• IP

Collaborator

Immunoprecipitation - Anti-ErbB2 / HER2 antibody [CAL27] (AB237715)

ERbB 2 was immunoprecipitated from 0.35 mg of HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab237715 at 130 dilution. Western blot was performed from the immunoprecipitate using ab237715 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used as secondary antibody at 1/5000 dilution.

Lane 1 : HeLa whole cell lysate 10 μg (Input).
Lane 2 : ab237715 IP in HeLa whole lysate.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab237715 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.
Exposure time : 15 seconds.

All lanes:

Immunoprecipitation - Anti-ErbB2 / HER2 antibody [CAL27] (ab237715)

Predicted band size: 137 kDa

Observed band size: 180 kDa

false

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ErbB2 / HER2 antibody [CAL27] (AB237715)

Immunohistochemical analysis of mouse breast carcinoma tissue labeling ErbB2 / HER2 with ab237715 at 1/2000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on the human breast carcinoma is observed. Counter stained with hematoxylin. Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins. The section was incubated with ab237715 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

• WB

Supplier Data

Western blot - Anti-ErbB2 / HER2 antibody [CAL27] (AB237715)

False colour image of Western blot : Anti-ErbB2 / HER2 antibody [CAL27] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab237715 was shown to bind specifically to ErbB2 / HER2. A band was observed at 180 kDa in wild-type A549 cell lysates with no signal observed at this size in ERBB2 knockout cell line. To generate this image, wild-type and ERBB2 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-ErbB2 / HER2 antibody [CAL27] (ab237715) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

ERBB2 knockout A549 cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

SK-BR-3 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 137 kDa

Observed band size: 180 kDa

false

• WB

Supplier Data

Western blot - Anti-ErbB2 / HER2 antibody [CAL27] (AB237715)

Blocking and dilution buffer : 5% NFDM/TBST.

This blot was developed using a higher sensitivity ECL substrate.

All lanes:

Western blot - Anti-ErbB2 / HER2 antibody [CAL27] (ab237715) at 1/1000 dilution

All lanes:

HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate 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 1/100000 dilution

Predicted band size: 137 kDa

Observed band size: 180 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-ErbB2 / HER2 antibody [CAL27] (AB237715)

False colour image of Western blot : Anti-ErbB2 / HER2 antibody [CAL27] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab237715 was shown to bind specifically to ErbB2 / HER2. A band was observed at 180 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in ERBB2 knockout cell line. To generate this image, wild-type and ERBB2 knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-ErbB2 / HER2 antibody [CAL27] (ab237715) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

ERBB2 knockout HCT 116 cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

SK-BR-3 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 137 kDa

Observed band size: 180 kDa

false

• WB

Lab

Western blot - Anti-ErbB2 / HER2 antibody [CAL27] (AB237715)

Anti-ErbB2 / HER2 antibody [CAL27] staining at 1/500 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab237715 was shown to bind specifically to ErbB2 / HER2. A band was observed at 180 kDa in wild-type MCF7 cell lysates with no signal observed at this size in ERBB2 knockout cell line ab286260 (knockout cell lysate ab300208). To generate this image, wild-type and ERBB2 knockout MCF7 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were HRP conjugated Goat anti-Rabbit (H+L) and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-ErbB2 / HER2 antibody [CAL27] (ab237715) at 1/500 dilution

Lane 1:

Wild-type MCF7 cell lysate at 32 µg

Lane 2:

Western blot - Human ErbB2 / HER2 knockout MCF7 cell line (<a href='/en-us/products/cell-lines/human-erbb2-her2-knockout-mcf7-cell-line-ab286260'>ab286260</a>)

Lane 2:

Western blot - Human ERBB2 knockout MCF7 cell lysate (ab300208)

Lane 2:

ERBB2 knockout MCF7 cell lysate at 32 µg

Lane 3:

SK-BR-3 cell lysate at 16 µg

Secondary

All lanes:

HRP conjugated Goat anti-Rabbit (H+L) and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 137 kDa

Observed band size: 180 kDa

false

• WB

Supplier Data

Western blot - Anti-ErbB2 / HER2 antibody [CAL27] (AB237715)

Blocking and dilution buffer : 5% NFDM/TBST.

This blot was developed using a higher sensitivity ECL substrate.

All lanes:

Western blot - Anti-ErbB2 / HER2 antibody [CAL27] (ab237715) at 1/1000 dilution

Lane 1:

4T1 (mouse mammary gland carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

C6 (rat glial tumor glial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Predicted band size: 137 kDa

Observed band size: 180 kDa

false

Exposure time: 3min