710213

antibody from Invitrogen Antibodies

Targeting: EGFR ERBB, ERBB1

Antibody data

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Validation data

Product number: 710213 - Provider product page
Provider: Invitrogen Antibodies
Product name: Phospho-EGFR (Tyr1148) Recombinant Polyclonal Antibody (8HCLC)
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: This antibody is predicted to react with equine, mouse, rabbit and rat based on sequence homology.
Antibody clone number: 8HCLC
Concentration: 0.5 mg/mL

EGFR protein structure - 710213 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Phospho-EGFR pTyr1148 in whole cell extracts of serum-starved A431 treated with 100 ng/mL of EGF for 5 min (lanes 1 and 2) using a Phospho-EGFR pTyr1148 Recombinant Rabbit Polyclonal Antibody (Product # 710213) at a dilution of 2 µg/mL. To confirm specificity, competition was performed by preincubation with the phosphopeptide to inhibit antibody binding (lane 2). Samples were detected using chemiluminescence (ECL). Results show a band at ~175 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (30 µg) of A549 (Lane 1), A431 (Lane2) and HeLa (Lane 3). The blots were probed with Recombinant Rabbit Polyclonal Phospho-EGFR pTyr1148 Antibody (Product # 710213, 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 ~ 180 kDa band corresponding to Phospho-EGFR pTyr1148 was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10 % Bis-Tris gel (Product # NP0302BOX), 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 Pierce™ Power Blotter System (22834). 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)
Main image
Experimental details: Western blot analysis of Phospho-EGFR pTyr1148 in whole cell extracts of serum-starved A431 treated with 100 ng/mL of EGF for 5 min (lanes 1 and 2) using a Phospho-EGFR pTyr1148 Recombinant Rabbit Polyclonal Antibody (Product # 710213) at a dilution of 2 µg/mL. To confirm specificity, competition was performed by preincubation with the phosphopeptide to inhibit antibody binding (lane 2). Samples were detected using chemiluminescence (ECL). Results show a band at ~175 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Phospho-EGFR pTyr1148 was performed using 70% confluent log phase A-431 cell treated with 20 ng 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 pTyr1148 (8HCLC) Recombinant Rabbit Polyclonal Antibody (Product # 710213) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature 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 Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing punctate cytoplasmic localization. Panel e is untreated cell with no signal. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Phospho-EGFR (Tyr1148) was performed using 90% 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 (Tyr1148) Recombinant Rabbit Polyclonal Antibody (Product # 710213) 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 membranous localization. Panel e represents cells treated with antagonist, Afatinib (1uM for 6hrs) followed by EGF (200 ng/mL for 10 minutes), showing reduced Phospho-EGFR (Tyr1148) 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.