Anti-EGFR (phospho Y1045) antibody [1HCLC]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-EGFR (phospho Y1045) antibody [1HCLC] (AB277783)

Immunofluorescence analysis of Phospho-EGFR (Tyr1045) was performed using 70% confluent log phase A-431 cells treated with 200 ng/mL of EGF for 10 minutes. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Phospho-EGFR (Tyr1045) Recombinant Rabbit Polyclonal Antibody (ab277783) at 2 μg/mL in 0.1% BSA and incubated overnight at +4°C and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal^™ Secondary Antibody, Alexa Fluor^® 488 conjugate at a dilution of 1/2000 for 45 minutes at room temperature (Panel a : green). Nuclei (Panel b : blue) were stained with SlowFade^® Gold Antifade Mountant with DAPI. F-actin (Panel c : red) was stained with Rhodamine Phalloidin (1/300). Panel d represents the merged image showing membrane localization. Panel e represents cells treated with antagonist, Afatinib (1uM for 6hrs) followed by EGF (200 ng/mL for 10 minutes), showing no Phospho-EGFR staining. Panel f shows untreated cells with no signal. Panel g represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-EGFR (phospho Y1045) antibody [1HCLC] (AB277783)

For immunofluorescence analysis A431 cells were fixed and permeabilized for detection of EGFR pY1045 using Anti-EGFR pY1045 Recombinant Rabbit Multiclonal Antibody (ab277783, 2 μ/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal^™ Secondary Antibody, Alexa Fluor^® 488 conjugate (1/2000). Nuclei (blue) were stained using SlowFade^® Gold Antifade Mountant with DAPI, and Alexa Fluor^® 555 Rhodamine Phalloidin (1/300) was used for cytoskeletal F-actin (red) staining. Detection and localization of EGFR pY1045 (green) in the membrane/cytoplasm can be clearly observed in cells treated with EGF (100 ng/mL, 15 min) as compared to untreated cells. Antibody specificity was demonstrated by competition with the EGFR pY1045 peptide, which results in loss of signal. No competition was observed with the non-phospho peptide. The images were captured at 60X magnification.

• WB

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Western blot - Anti-EGFR (phospho Y1045) antibody [1HCLC] (AB277783)

All lanes:

Western blot - Anti-EGFR (phospho Y1045) antibody [1HCLC] (ab277783) at 2 µg/mL

Lane 1:

A431 (human epidermoid carcinoma cell line) whole cell extract

Lane 2:

A431 (human epidermoid carcinoma cell line) whole cell extract (treated with EGF 10 ng / mL for 5 minutes)

Predicted band size: 134 kDa

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