- 711368 - Invitrogen Antibodies EGFR antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of A-431 (Lane 1) and A-431 treated with EGF (10 ng/mL for 5 min) (Lane 2). The blots were probed with Anti-EGFR (pY1045) Recombinant Rabbit Polyclonal Antibody (Product # 711368, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 175 kDa band corresponding to EGFR (pY1045) was observed. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with overnight wet transfer system. The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: For immunofluorescence analysis A431 cells were fixed and permeabilized for detection of EGFR pY1045 using Anti-EGFR pY1045 Recombinant Rabbit Polyclonal Antibody (Product # 711368, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Nuclei (blue) were stained using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938), and Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 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 (Product # 711368) at 2 µg/mL in 0.1% BSA and incubated overnight at 4 degree Celsius and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) 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 (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 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.

711368