Anti-EGFR (phospho Y1148) antibody [RP23040050]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-EGFR (phospho Y1148) antibody [RP23040050] (AB307989)

For immunofluorescence analysis, 90% confluent log phase A-431 cells treated with 200 ng/mL of EGF for 10 minutes 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 for detection of EGFR (phospho Y1148) using ab307989 at 2 μg/mL in 0.1% BSA and incubated overnight at 4degC and then labeled with Goat anti-Rabbit IgG (H+L) 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 DAPI. F-actin (Panel c : red) was stained with Rhodamine Phalloidin at 1/300. Panel d represents the merged image showing membranous localization. Panel e represents cells treated with antagonist, Afatinib (1μM for 6 hours) followed by EGF (200 ng/mL for 10 minutes), showing reduced EGFR (phospho Y1148) 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

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-EGFR (phospho Y1148) antibody [RP23040050] (AB307989)

For immunofluorescence analysis, 70% confluent log phase A-431 cell treated with 20 ng of EGF for 10 minutes 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 for detection of EGFR (phospho Y1148) using ab307989 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) 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 DAPI. F-actin (Panel c : red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin at 1/300 dilution. 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.

• WB

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Western blot - Anti-EGFR (phospho Y1148) antibody [RP23040050] (AB307989)

Western blot analysis of EGFR (phospho Y1148) in whole cell extracts of serum-starved A431 treated with 100 ng/mL of EGF for 5 min (lanes 1 and 2) using ab307989 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 around 175 kDa.

All lanes:

Western blot - Anti-EGFR (phospho Y1148) antibody [RP23040050] (ab307989) at 2 µg/mL

Lane 1:

EGF treated whole cell extracts of serum-starved A431 cells without phosphopeptide

Lane 2:

EGF treated whole cell extracts of serum-starved A431 cells with phosphopeptide

Observed band size: 175 kDa

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• WB

Supplier Data

Western blot - Anti-EGFR (phospho Y1148) antibody [RP23040050] (AB307989)

Western blot analysis was performed on whole cell extracts of A549, A431 and HeLa. The blots were probed with ab307989 at 2 µg/mL dilution and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP conjugate at 1/2500 dilution (0.4 µg/mL). A approximate 180 kDa band corresponding to EGFR (phospho Y1148) was observed across cell lines tested. Known quantity of protein samples were electrophoresed using 10 % Bis-Tris gel, electrophoresis system and pre-stained protein standard. Resolved proteins were then transferred onto a nitrocellulose membrane. The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was then performed.

All lanes:

Western blot - Anti-EGFR (phospho Y1148) antibody [RP23040050] (ab307989) at 2 µg/mL

Lane 1:

A549 cells at 30 µg

Lane 2:

A431 cells at 30 µg

Lane 3:

HeLa cells at 30 µg

Secondary

All lanes:

HRP-conjugated Goat anti-Rabbit IgG (H+L) Secondary Antibody at 1/2500 dilution

Observed band size: 180 kDa

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