Anti-ErbB2 / HER2 antibody [EPR19547-12] - BSA and Azide free

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-ErbB2 / HER2 antibody [EPR19547-12] - BSA and Azide free (AB222482)

Flow cytometry overlay histogram showing T-47D cells stained with ab214275 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab214275) (1x 10⁶ in 100μl at a 1/2500 dilution for 30 min at 22° C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed was incubated at a 1/4000 dilution for 30 min at 22° C.

Isotype control antibody was (black line) ab172730 Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW blue laser (488nm) and 525/40 bandpass filter.

This antibody gave a positive signal in T-47D fixed with 80% methanol (5 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab214275).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-ErbB2 / HER2 antibody [EPR19547-12] - BSA and Azide free (AB222482)

Immunofluorescent analysis of 100% methanol-fixed SK-BR-3 (Human mammary gland adenocarcinoma cell line) cells labeling ErbB2 / HER2 with ab214275 at 1/250 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing membrane staining on SK-BR-3 cells.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (red).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab214275).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ErbB2 / HER2 antibody [EPR19547-12] - BSA and Azide free (AB222482)

Immunohistochemical analysis of paraffin-embedded Human breast tissue labeling ErbB2 / HER2 with ab214275 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Negative control : No staining on normal Human breast [PMID : 15150568].

Counter stained with Hematoxylin.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab214275).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ErbB2 / HER2 antibody [EPR19547-12] - BSA and Azide free (AB222482)

Immunohistochemical analysis of paraffin-embedded Human breast cancer tissue labeling ErbB2 / HER2 with ab214275 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Membrane staining on Human breast cancer is observed [PMID : 18437174].

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab214275).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-ErbB2 / HER2 antibody [EPR19547-12] - BSA and Azide free (AB222482)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed SK-BR-3 (Human mammary gland adenocarcinoma cell line) cells labeling ErbB2 / HER2 with ab214275 at 1/60 dilution (red) compared with a RabbitIgG,monoclonal [EPR25A] - Isotype control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti Rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab214275).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ErbB2 / HER2 antibody [EPR19547-12] - BSA and Azide free (AB222482)

Immunohistochemical analysis of paraffin-embedded Human breast cancer tissue labeling ErbB 2 with ab214275 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Membrane staining on Human breast cancer is observed [PMID : 18437174].

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab214275).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

Supplier Data

Immunoprecipitation - Anti-ErbB2 / HER2 antibody [EPR19547-12] - BSA and Azide free (AB222482)

ErbB2 / HER2 was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab214275 at 1/30 dilution.

Western blot was performed from the immunoprecipitate using ab214275 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1 : HeLa whole cell lysate 10μg (Input).

Lane 2 : ab214275 IP in HeLa whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab214275 in HeLa whole cell lysate.

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

Exposure time : 3 seconds.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab214275).

All lanes:

Immunoprecipitation - Anti-ErbB2 / HER2 antibody [EPR19547-12] (<a href='/en-us/products/primary-antibodies/erbb2-her2-antibody-epr19547-12-ab214275'>ab214275</a>)

Predicted band size: 137 kDa

false

• I-ELISA

Lab

Indirect ELISA - Anti-ErbB2 / HER2 antibody [EPR19547-12] - BSA and Azide free (AB222482)

This data was developed using the same antibody clone in a different buffer formulation (ab222482).

ELISA using ab214275 at using 1000 ng/ml an antigen concentration range of 500-0 ng/ml for Human ERBB2 and ERBB4 Recombinant Proteins. Goat Anti-Rabbit IgG (HRL) (1/50000) was used as the secondary antibody. 3,3',5,5'-Tetramethylbenzidine (TMB) used as substrate solution.

• WB

Supplier Data

Western blot - Anti-ErbB2 / HER2 antibody [EPR19547-12] - BSA and Azide free (AB222482)

This data was developed using the same antibody clone in a different buffer formulation (ab214275). Blocking/Dilution buffer : 5% NFDM/TBST. Exposure time : Lane 1 : 3 minutes; Lane 2 : 3 seconds; Lane 3 : 10 seconds. The molecular weight observed is consistent with the literature PMID : 15741050.

All lanes:

Western blot - Anti-ErbB2 / HER2 antibody [EPR19547-12] (<a href='/en-us/products/primary-antibodies/erbb2-her2-antibody-epr19547-12-ab214275'>ab214275</a>) at 1/1000 dilution

Lane 1:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

Sample: A431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 3:

SK-BR-3 (Human mammary gland adenocarcinoma cell line) 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

false

• WB

Supplier Data

Western blot - Anti-ErbB2 / HER2 antibody [EPR19547-12] - BSA and Azide free (AB222482)

This data was developed using the same antibody clone in a different buffer formulation (ab214275). Blocking/Dilution buffer : 5% NFDM/TBST. The molecular weight is consist with literature PMID : 15741050.

All lanes:

Western blot - Anti-ErbB2 / HER2 antibody [EPR19547-12] (<a href='/en-us/products/primary-antibodies/erbb2-her2-antibody-epr19547-12-ab214275'>ab214275</a>) at 1/2000 dilution

All lanes:

Human breast cancer lysate at 10 µg

false

• WB

Supplier Data

Western blot - Anti-ErbB2 / HER2 antibody [EPR19547-12] - BSA and Azide free (AB222482)

This data was developed using the same antibody clone in a different buffer formulation (ab214275). Blocking/Dilution buffer : 5% NFDM/TBST. The molecular weight is consist with literature PMID : 15741050.

All lanes:

Western blot - Anti-ErbB2 / HER2 antibody [EPR19547-12] (<a href='/en-us/products/primary-antibodies/erbb2-her2-antibody-epr19547-12-ab214275'>ab214275</a>) at 10 µg

Lane 1:

C6 (Rat glial tumor cell line) whole cell lysate at 10 µg

Lane 2:

PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 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

false

• WB

Supplier Data

Western blot - Anti-ErbB2 / HER2 antibody [EPR19547-12] - BSA and Azide free (AB222482)

This data was developed using the same antibody clone in a different buffer formulation (ab214275). Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-ErbB2 / HER2 antibody [EPR19547-12] (<a href='/en-us/products/primary-antibodies/erbb2-her2-antibody-epr19547-12-ab214275'>ab214275</a>)

All lanes:

4T1 (Mouse mammary gland carcinoma epithelial cell) 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 1/20000 dilution

false

• WB

Lab

Western blot - Anti-ErbB2 / HER2 antibody [EPR19547-12] - BSA and Azide free (AB222482)

This data was developed using the same antibody clone in a different buffer formulation (ab222482). Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 180 seconds. This antibody could recognize ErbB2 and weak cross reaction with ErbB4.

Lanes 1 - 2:

Western blot - Anti-ErbB2 / HER2 antibody [EPR19547-12] (<a href='/en-us/products/primary-antibodies/erbb2-her2-antibody-epr19547-12-ab214275'>ab214275</a>) at 1/1000 dilution

Lanes 1 - 2:

Western blot - Anti-ErbB2 / HER2 antibody [EPR19547-12] - BSA and Azide free (ab222482) at 1/1000 dilution

Lane 1:

His-tagged Recombinant Human ErbB2 protein (aa676-1255)

Lane 2:

His-tagged Recombinant Human ErbB4 protein (aa676-1308)

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: 137 kDa

Observed band size: 90 kDa,80 kDa

false