Anti-EGFR antibody [E235]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-EGFR antibody [E235] (AB32077)

Immunocytochemistry/Immunofluorescence analysis of A431 cells labelling EGFR with ab32077 at 1/500. Cells were fixed with 100% methanol. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody.
Control : PBS only.
Nuclear counter stain : DAPI.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-EGFR antibody [E235] (AB32077)

Overlay histogram showing A431 cells stained with ab32077 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32077, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

• IP

Unknown

Immunoprecipitation - Anti-EGFR antibody [E235] (AB32077)

Purified ab32077 at 1/50 dilution (2μg) immunoprecipitating EGFR in A431 whole cell lysate.
Lane 1 (input) : A431 (Human epidermoid carcinoma epithelial cell) whole cell lysate 10μg
Lane 2 (+) : ab32077 + A431 whole cell lysate.
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab32077 in A431 whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration : 5% NFDM/TBST.
Diluting buffer and concentration : 5% NFDM/TBST.
Observed band size : 180 kDa

All lanes:

Immunoprecipitation - Anti-EGFR antibody [E235] (ab32077)

Predicted band size: 134 kDa

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• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [E235] (AB32077)

IHC image of EGFR staining in a formalin fixed, paraffin embedded mouse normal skin tissue section, performed on a Leica Bond™ system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab32077 at 1/200 dilution for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

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.

• WB

Lab

Western blot - Anti-EGFR antibody [E235] (AB32077)

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with ab32077 overnight at 4°C in the presence of loading control ab18058 (Mouse monoclonal [SPM227] to Vinculin diluted 1 : 10000). Antibody binding was detected using IR-labelled goat anti-Rabbit Ab at a 1 : 10,000 dilution for one hour at room temperature before imaging.

This product yielded a strong signal in western blot using A431 (human squamous carcinoma) lysate which naturally overexpresses the EGFR protein. Western blot conditions may need to be optimised for cell lines and tissues that express lower levels of endogenous EGFR.

All lanes:

Western blot - Anti-EGFR antibody [E235] (ab32077) at 1/1000 dilution

Lane 1:

Caco-2 cell lysate at 20 µg

Lane 2:

A431 cell lysate at 20 µg

Lane 3:

Mouse skin cell lysate at 20 µg

Lane 4:

Rat skin cell lysate at 20 µg

Predicted band size: 134 kDa

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• WB

Supplier Data

Western blot - Anti-EGFR antibody [E235] (AB32077)

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-EGFR antibody - Total protein control (ab32077) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-EGFR (phospho Y1045) antibody [EPR28381-88] (<a href='/en-us/products/primary-antibodies/egfr-phospho-y1045-antibody-epr28381-88-ab316155'>ab316155</a>) at 1/1000 dilution

Lane 1:

Untreated A431 (human epidermoid carcinoma epithelial cell) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

A431 treated with 100 ng/ml EGF for 30 minutes whole cell lysate (untreated membrane) at 20 µg

Lane 3:

A431 treated with 100 ng/ml EGF for 30 minutes whole cell lysate (alkaline phosphatase treated membrane) 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

Observed band size: 175 kDa,36 kDa

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Exposure time: 10s

• WB

Lab

Western blot - Anti-EGFR antibody [E235] (AB32077)

Western blot : Anti-EGFR antibody [E235] (ab32077) 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, ab32077 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 [E235] (ab32077) 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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• WB

Supplier Data

Western blot - Anti-EGFR antibody [E235] (AB32077)

In Western blot, ab316155 was shown to bind specifically to EGFR (phospho Y1045). Target of interest was observed at 175 kDa in wild-type HeLa cell lysates (lane 2) with no signal observed at this size in EGFR knockout cell line (lanes 3-4 KO cell line ab255385/KO cell lysate ab263845).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-EGFR antibody (ab32077) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-EGFR (phospho Y1045) antibody [EPR28381-88] (<a href='/en-us/products/primary-antibodies/egfr-phospho-y1045-antibody-epr28381-88-ab316155'>ab316155</a>) at 1/1000 dilution

Lane 1:

Untreated Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Wild-type HeLa treated with 100 ng/ml EGF for 30 minutes whole cell lysate at 20 µg

Lane 3:

Untreated EGFR knockout HeLa whole cell lysate at 20 µg

Lane 4:

EGFR knockout HeLa treated with 100 ng/ml EGF for 30 minutes 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

Observed band size: 175 kDa,36 kDa

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Exposure time: 180s

• WB

Lab

Western blot - Anti-EGFR antibody [E235] (AB32077)

** Lanes 1 - 4 : ** Merged signal (red and green). Green - ab32077 observed at 175 kDa. Red - loading control, ab130007 observed at 125 kDa. ab32077 was shown to react with EGFR in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab255385 (knockout cell lysate ab263845) was used. Wild-type and EGFR knockout samples were subjected to SDS-PAGE. ab32077 and Anti-Vinculin antibody [VIN-54] (ab130007) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-EGFR antibody [E235] (ab32077) at 1/1000 dilution

Lane 1:

A431 cell lysate at 20 µg

Lane 2:

MDA-MB-468 cell lysate at 20 µg

Lane 3:

Wild type HeLa cell lysate at 20 µg

Lane 4:

EGFR knockout HeLa cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Predicted band size: 134 kDa

Observed band size: 124 kDa,134 kDa

false

• WB

Lab

Western blot - Anti-EGFR antibody [E235] (AB32077)

False colour image of Western blot : Anti-EGFR antibody [E235] 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, ab32077 was shown to bind specifically to EGFR. A band was observed at 180 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in Egfr knockout cell line ab281597 (knockout cell lysate ab282949). To generate this image, wild-type and Egfr 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 (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-EGFR antibody [E235] (ab32077) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

EGFR knockout HCT 116 cell lysate at 20 µg

Lane 2:

Western blot - Human EGFR knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-egfr-knockout-hct116-cell-line-ab281597'>ab281597</a>)

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

Caco-2 cell lysate at 20 µg

Predicted band size: 134 kDa

Observed band size: 180 kDa

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