Anti-ErbB2 / HER2 antibody [CPT-R32.2-27-12]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-ErbB2 / HER2 antibody [CPT-R32.2-27-12] (AB241325)

Immunofluorescent analysis of 100% methanol fixed SK-BR-3 (human breast adenocarcinoma epithelial cell) cells labeling ErbB 2 with ab241325 at 1/50 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 cells.

Negative control : MCF7 (Human breast adenocarcinoma cell line) (PMID : 20697531). Negative staining was obtained from 4% PFA fixation.

The nuclear counter stain is DAPI (blue). Tubulin is detected with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at a 1/200 dilution (red).

• WB

Lab

Western blot - Anti-ErbB2 / HER2 antibody [CPT-R32.2-27-12] (AB241325)

False colour image of Western blot : Anti-ErbB2 / HER2 antibody [CPT-R32.2-27-12] 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, ab241325 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 [CPT-R32.2-27-12] (ab241325) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 40 µg

Lane 2:

ERBB2 knockout HeLa cell lysate at 40 µg

Predicted band size: 137 kDa

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• WB

Supplier Data

Western blot - Anti-ErbB2 / HER2 antibody [CPT-R32.2-27-12] (AB241325)

Negative control-MCF7. MCF7 belongs to Luminal A subtype which is HER2 negative (PMID : 20697531).

HER2 has 6 potential glycosylation sites, which might lead to the molecular mass greater than the predicted size. The expression profile in SK-BR-3 cells is consistent with literature (PMID : 24425042). The multiple bands likely represent degraded HER2 or different isoforms.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-ErbB2 / HER2 antibody [CPT-R32.2-27-12] (ab241325) at 1/1000 dilution

Lane 1:

SK-BR-3 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

MCF7 (human breast adenocarcinoma 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 1/100000 dilution

Predicted band size: 137 kDa

Observed band size: 150-250 kDa

false

Exposure time: 26s

• ELISA

Supplier Data

ELISA - Anti-ErbB2 / HER2 antibody [CPT-R32.2-27-12] (AB241325)

ELISA - Anti-ErbB 2 antibody [CPT-R32.2-27-12] (ab241325) used at 1000 ng/mL.

50 ng CPTAC-36d (1000 ng/mL, 50 μL per well) was coated onto 96-wells. Serial dilutions (0, 0.2, 0.9, 3.9, 15, 62, 250, 1000 ng/mL) of ab241325 (50 μL) were incubated with CPTAC-36d in each well for 60 minutes by shaking. This was followed by adding goat anti-rabbit IgG, (H+L), phosphatase-conjugated secondary antibody (50 μL, 1/2500) into each well and incubating for another 40 minutes. After washing, 50 μL of PNPP was added and incubated for 15 minutes without shaking for color development. OD was read at 405nm within 5 minutes.

Primary antibody concentration range : 0.01 - 1.0 μg/mL.