Anti-EGFR (phospho Y1068) antibody

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-EGFR (phospho Y1068) antibody (AB5644)

Immunofluorescence analysis of A431 (Human epidermoid carcinoma cell line) cells labeling EGFR with ab5644 at 1/100 dilution, followed by Goat anti-rabbit IgG (H+L) Superclonal, Alexa Fluor® 488 conjugate was used as the secondary antibody at 1/2000 dilution (Panel a). Nuclei (Panel b) were stained with SlowFade^® Gold Antifade Mountant with DAPI. F-actin (Panel c) was stained with Rhodamine Phalloidin. Panel (d) represents the merged image showing membrane localization. Panel (e) represents cells treated with antagonist, Afatinib (1μM 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 cells (in 70% confluent log phase treated with 200ng/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 hour at room temperature. The images were captured at 60X magnification.

• WB

Supplier Data

Western blot - Anti-EGFR (phospho Y1068) antibody (AB5644)

Western blot analysis using ab5644 shows increased expression of proteins phosphorylated at the tyrosine residues in A-431 and A549 cell lines upon EGF treatment and pre-treatment with EGFR-antagonists, Gefitinib and Afatinib. This results in inhibition of Phospho-EGFR in A-431 and A549 cell lines

All lanes:

Western blot - Anti-EGFR (phospho Y1068) antibody (ab5644) at 1/1000 dilution

Lane 1:

A431 (Human epidermoid carcinoma cell line) whole cell lysate at 30 µg

Lane 2:

A431 whole cell lysate treated with EGF (200 ng/mL for 10 minutes) at 30 µg

Lane 3:

A431 whole cell lysate treated with Gefitinib followed by EGF (1uM for 16 hours, 200 ng/mL for 10 minutes) at 30 µg

Lane 4:

A431 whole cell lysate treated with Afatinib followed by EGF (0.5 uM for 6 hours, 200 ng/mL for 10 minutes) at 30 µg

Lane 5:

A549 (Human lung carcinoma cell line) whole cell lysate at 30 µg

Lane 6:

A549 whole cell lysate treated with EGF (200 ng/mL for 10 minutes) at 30 µg

Lane 7:

A549 whole cell lysate treated with Afatinib followed by EGF (0.5 uM for 6 hours, 200 ng/mL for 10 minutes) at 30 µg

Secondary

All lanes:

Goat anti-Rabbit IgG Superclonal Secondary Antibody, HRP at 1/4000 dilution

Predicted band size: 134 kDa

false

• WB

AbReview5553****

Western blot - Anti-EGFR (phospho Y1068) antibody (AB5644)

All lanes:

Western blot - Anti-EGFR (phospho Y1068) antibody (ab5644) at 1/200 dilution

Lane 1:

A431 (Human epidermoid carcinoma cell line) whole cell lysate - not treated

Lane 2:

A431 whole cell lysate - 100ng/ml EGF for 1 minute

Lane 3:

A431 whole cell lysate - 100ng/ml EGF for 2.5 minutes

Lane 4:

A431 whole cell lysate - 100ng/ml EGF for 5 minutes

Lane 5:

A431 whole cell lysate - 100ng/ml EGF for 10 minutes

Lane 6:

A431 whole cell lysate - 100ng/ml EGF for 20 minutes

Lane 7:

A431 whole cell lysate - 100ng/ml EGF for 40 minutes

Secondary

All lanes:

HRP conjugated Goat anti-rabbit antibody

Predicted band size: 134 kDa

true

Exposure time: 2min

This image is courtesy of an Abreview submitted by Mr Samir Nuseibeh