Antibody data
- Antibody Data
- Antigen structure
- References [0]
- Comments [0]
- Validations
- Western blot [2]
- Immunocytochemistry [1]
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Validation data
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- Product number
- PA5-17848 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Phospho-EGFR (Tyr1068) Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Description
- It is not recommended to aliquot this antibody.
- Reactivity
- Human, Mouse, Rat
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 100 µL
- Concentration
- 5 µg/mL
- Storage
- -20°C
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis was performed on membrane enriched extracts (30 µg lysate) of A-431 (Lane 1), A-431 treated with EGF (200 ng/mL for 10 minutes) (Lane 2), A-431 treated with Gefitinib followed by EGF (1uM for 16 hours, 200 ng/mL for 10 minutes) (Lane 3), A-431 treated with Afatinib followed by EGF (0.5 uM for 6 hours, 200 ng/mL for 10 minutes) (Lane 4), A549 (Lane 5), A549 treated with EGF (200 ng/mL for 10 minutes) (Lane 6) and A549 treated with Afatinib followed by EGF (0.5 uM for 6 hours, 200 ng/mL for 10 minutes) (Lane 7). The blot was probed with Anti-Phospho-EGFR (Tyr1068) Rabbit Polyclonal Antibody (Product # PA5-17848, 1:1000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 170 kDa band corresponding to Phospho-EGFR (Tyr1068) was detected and observed to increase upon EGF treatment across cell lines tested. Pre-treatment with EGFR-antagonists, Gefitinib and Afatinib, resulted in inhibition of Phospho-EGFR in A-431 and A549 cell lines. 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 tra
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis was performed on membrane enriched extracts (30 µg lysate) of A-431 (Lane 1), A-431 treated with EGF (200 ng/mL for 10 minutes) (Lane 2), A-431 treated with Gefitinib followed by EGF (1uM for 16 hours, 200 ng/mL for 10 minutes) (Lane 3), A-431 treated with Afatinib followed by EGF (0.5 uM for 6 hours, 200 ng/mL for 10 minutes) (Lane 4), A549 (Lane 5), A549 treated with EGF (200 ng/mL for 10 minutes) (Lane 6) and A549 treated with Afatinib followed by EGF (0.5 uM for 6 hours, 200 ng/mL for 10 minutes) (Lane 7). The blot was probed with Anti-Phospho-EGFR (Tyr1068) Rabbit Polyclonal Antibody (Product # PA5-17848, 1:1000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 170 kDa band corresponding to Phospho-EGFR (Tyr1068) was detected and observed to increase upon EGF treatment across cell lines tested. Pre-treatment with EGFR-antagonists, Gefitinib and Afatinib, resulted in inhibition of Phospho-EGFR in A-431 and A549 cell lines. 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 tra
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescence analysis of Phospho-EGFR (Tyr1068) 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 (Tyr1068) Rabbit Polyclonal Antibody (Product # PA5-17848) at 1:100 dilution in 0.1% BSA and incubated overnight at 4 degree 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.