Anti-EGFR (phospho Y1068) antibody (ab5644)

  Western blot - EGFR (phospho Y1068) antibody (ab5644)

Peptide Competition: Extracts prepared from NIH3T3 cells expressing EGFR were starved for 30 hours, then stimulated for 10 minutes with 30 ng/mL EGF (+), or left unstimulated (-), then resolved by SDS-PAGE on a 6% Tris-glycine gel, and transferred to nitrocellulose. Membranes were blocked with a 5% BSA-TBST buffer overnight at 4°C, then were incubated with 0.50 µg/mL ab5644 antibody, following prior incubation with: no peptide (1, 2), the phosphopeptide immunogen (3, 4), or, the non phosphopeptide corresponding to the phosphopeptide immunogen (5, 6). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and bands were detected using the Tropix WesternStar method. The data show that only the phosphopeptide corresponding to this site blocks the antibody signal, demonstrating the specificity of the ab5644 antibody for this phosphorylated residue. The data also show the activation of the EGFR after stimulation with EGF.

  Western blot - EGFR (phospho Y1068) antibody (ab5644)

All lanes : Anti-EGFR (phospho Y1068) antibody (ab5644) at 1/200 dilution

Lane 1 : A431 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
HRP conjugated Goat anti-rabbit antibody
developed using the ECL technique

Performed under reducing conditions.

Exposure time : 2 minutes

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

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