Anti-EGFR antibody [E235] - BSA and Azide free

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-EGFR antibody [E235] - BSA and Azide free (AB227459)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32077).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-EGFR antibody [E235] - BSA and Azide free (AB227459)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32077).

• IP

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Immunoprecipitation - Anti-EGFR antibody [E235] - BSA and Azide free (AB227459)

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
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32077).

All lanes:

Immunoprecipitation - Anti-EGFR antibody [E235] (<a href='/en-us/products/primary-antibodies/egfr-antibody-e235-ab32077'>ab32077</a>)

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] - BSA and Azide free (AB227459)

This IHC data was generated using the same anti-EGFR antibody clone, E235, in a different buffer formulation (cat# 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] - BSA and Azide free (AB227459)

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] (<a href='/en-us/products/primary-antibodies/egfr-antibody-e235-ab32077'>ab32077</a>) 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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• WB

Lab

Western blot - Anti-EGFR antibody [E235] - BSA and Azide free (AB227459)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32077).

All lanes:

Western blot - Anti-EGFR antibody [E235] (<a href='/en-us/products/primary-antibodies/egfr-antibody-e235-ab32077'>ab32077</a>) 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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