Anti-EGFR antibody [E234]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-EGFR antibody [E234] (AB32198)

Immunocytochemistry/ Immunofluorescence analysis of A431 (Human epidermoid carcinoma epithelial cell) cells labeling EGFR with purified ab32198 at 1/100 dilution (2.2 μg/mL). Cells were fixed in 100% Methanol. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 μg/mL). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 μg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-EGFR antibody [E234] (AB32198)

Intracellular Flow Cytometry analysis of A431 (Human epidermoid carcinoma epithelial cell) cells labeling EGFR with purified ab32198 at 1/200 dilution (10μg/mL) (Red). Cells were fixed with 80% Methanol and permeabilised with 0.1% Tween-20. A Goat anti rabbit IgG (Alexa Fluorr^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• WB

Lab

Western blot - Anti-EGFR antibody [E234] (AB32198)

Blocking/Diluting Buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-EGFR antibody [E234] (ab32198) at 1/1000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Lane 2:

A431 (Human epidermoid carcinoma epithelial cell) whole cell lysates 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

Predicted band size: 134 kDa

Observed band size: 150 kDa

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• WB

Lab

Western blot - Anti-EGFR antibody [E234] (AB32198)

Western blot : Anti-EGFR antibody [E234] (ab32198) 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, ab32198 was shown to bind specifically to EGFR. A band was observed at 160 kDa in wild-type cell lysates with no signal observed at this size in EGFR knockout cell lines. To generate this image, wild-type and EGFR knockout A549 (ab286394) and HeLa (ab255385) 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-EGFR antibody [E234] (ab32198) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

Western blot - Human EGFR knockout A549 cell line (<a href='/en-us/products/cell-lines/human-egfr-knockout-a549-cell-line-ab286394'>ab286394</a>)

Lane 2:

EGFR knockout A549 cell lysate at 20 µg

Lanes 2 and 4:

Western blot - Human EGFR knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-egfr-knockout-hela-cell-line-ab255385'>ab255385</a>)

Lane 3:

Wild-type HeLa cell lysate at 20 µg

Lane 4:

EGFR knockout HeLa cell lysate at 20 µg

Observed band size: 160 kDa

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