Anti-ErbB2 / HER2 antibody [EP1045Y] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ErbB2 / HER2 antibody [EP1045Y] - BSA and Azide free (AB194979)

This data was developed using ab134182, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of formalin fixed paraffin embedded human breast carcinoma labelling ErbB2/HER2 with ab134182 at a concentration of 0.1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab134182 anti-ErbB2 / HER2 antibody [EP1045Y]antibody was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ErbB2 / HER2 antibody [EP1045Y] - BSA and Azide free (AB194979)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human breast carcinoma tissue sections labeling ErbB2 / HER2 with Purified ab134182 at 1 : 1600 dilution (0.68 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1 : 0 dilution. PBS instead of the primary antibody was used as the negative control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134182).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-ErbB2 / HER2 antibody [EP1045Y] - BSA and Azide free (AB194979)

Immunofluorescent analysis of SKBR cells labelling ErbB2 / HER2 with ab134182 at 1/250 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134182).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-ErbB2 / HER2 antibody [EP1045Y] - BSA and Azide free (AB194979)

Immunocytochemistry/Immunofluorescence analysis of SK-BR-3 (human mammary gland adenocarcinoma) labelling ErbB2 / HER2 with purified ab134182 at 1/125. Cells were fixed with 4% PFA and permeabilized with 0.1% Triton X-100. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody (ab150077). Nuclei counterstained with DAPI (blue).

Control : PBS only

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134182).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ErbB2 / HER2 antibody [EP1045Y] - BSA and Azide free (AB194979)

Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labelling ErbB2 / HER2 with ab134182 at 1/100 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134182).

Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.

• IP

Unknown

Immunoprecipitation - Anti-ErbB2 / HER2 antibody [EP1045Y] - BSA and Azide free (AB194979)

ab134182 (purified) at 1 : 30 dilution (2μg) immunoprecipitating ErbB2 / HER2 in HeLa whole cell lysate.
Lane 1 (input) : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10μg
Lane 2 (+) : ab134182 & HeLa whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab134182 in HeLa whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1 : 1000 dilution.
Blocking and diluting buffer : 5% NFDM/TBST.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134182).

All lanes:

Immunoprecipitation - Anti-ErbB2 / HER2 antibody [EP1045Y] (<a href='/en-us/products/primary-antibodies/erbb2-her2-antibody-ep1045y-ab134182'>ab134182</a>)

Predicted band size: 137 kDa

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• WB

Lab

Western blot - Anti-ErbB2 / HER2 antibody [EP1045Y] - BSA and Azide free (AB194979)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134182). Western blot : Anti-ErbB2 / HER2 antibody [EP1045Y] staining at 1/500 dilution, shown in black; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab134182 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 and washed again four times. Secondary antibodies used were HRP conjugated Goat anti-Rabbit (H+L) and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution. This blot was developed with an ultra high-sensitivity ECL substrate kit and imaged with 20 minutes exposure time.

All lanes:

Western blot - Anti-ErbB2 / HER2 antibody [EP1045Y] (<a href='/en-us/products/primary-antibodies/erbb2-her2-antibody-ep1045y-ab134182'>ab134182</a>) at 1/500 dilution

Lane 1:

Wild-type MCF7 cell lysate at 32 µg

Lane 2:

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

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:

ERBB2 knockout MCF7 cell lysate at 32 µg

Lane 3:

A549 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

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• WB

Lab

Western blot - Anti-ErbB2 / HER2 antibody [EP1045Y] - BSA and Azide free (AB194979)

Blocking buffer and concentration : 5% NFDM/TBST

Diluting buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-ErbB2 / HER2 antibody [EP1045Y] - BSA and Azide free (ab194979)

All lanes:

SKBR-3 (human mammary gland adenocarcinoma) 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>)

Predicted band size: 137 kDa

Observed band size: 185 kDa

false

Exposure time: 3s

• WB

Lab

Western blot - Anti-ErbB2 / HER2 antibody [EP1045Y] - BSA and Azide free (AB194979)

False colour image of Western blot : Anti-ErbB2 / HER2 antibody [EP1045Y] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab134182 was shown to bind specifically to ErbB2 / HER2. A band was observed at 180 kDa in wild-type HeLa cell lysates with no signal observed at this size in ERBB2 knockout cell line ab255387 (knockout cell lysate ab263758). To generate this image, wild-type and ERBB2 knockout HeLa 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 (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-ErbB2 / HER2 antibody [EP1045Y] (<a href='/en-us/products/primary-antibodies/erbb2-her2-antibody-ep1045y-ab134182'>ab134182</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

ERBB2 knockout HeLa cell lysate at 20 µg

Predicted band size: 137 kDa

false

• WB

Supplier Data

Western blot - Anti-ErbB2 / HER2 antibody [EP1045Y] - BSA and Azide free (AB194979)

False colour image of Western blot : Anti-ErbB2 / HER2 antibody [EP1045Y] 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, ab134182 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. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134182).

All lanes:

Western blot - Anti-ErbB2 / HER2 antibody [EP1045Y] (<a href='/en-us/products/primary-antibodies/erbb2-her2-antibody-ep1045y-ab134182'>ab134182</a>) 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

Supplier Data

Western blot - Anti-ErbB2 / HER2 antibody [EP1045Y] - BSA and Azide free (AB194979)

False colour image of Western blot : Anti-ErbB2 / HER2 antibody [EP1045Y] 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, ab134182 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. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134182).

All lanes:

Western blot - Anti-ErbB2 / HER2 antibody [EP1045Y] (<a href='/en-us/products/primary-antibodies/erbb2-her2-antibody-ep1045y-ab134182'>ab134182</a>) 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

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-ErbB2 / HER2 antibody [EP1045Y] - BSA and Azide free (AB194979)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.