Phospho-EGFR (Y1148) Rabbit Polyclonal Antibody

Western blot analysis of Phospho-EGFR (Y1148) on different lysates with Rabbit anti-Phospho-EGFR (Y1148) antibody (HA500580) at 1/5,000 dilution.

Lane 1: A431 cell lysate
Lane 2: A431 starved overnight then treated with 20ng/mL EGF for 5 minutes cell lysate
Lane 3: A431 starved overnight then treated with 20ng/mL EGF for 5 minutes cell lysate, then the membrane treated with λpp for 1 hour

Lysates/proteins at 20 µg/Lane.

Predicted band size: 134 kDa
Observed band size: 170 kDa

Exposure time: 1 minute 23 seconds; ECL: K1801;
4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA500580) at 1/5,000 dilution was used in primary antibody dilution (K1803) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Immunocytochemistry analysis of A431 cells untreated / starved overnight then treated with 20ng/mL EGF for 5 minutes labeling Phospho-EGFR (Y1148) with Rabbit anti-Phospho-EGFR (Y1148) antibody (HA500580) at 1/10,000 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-EGFR (Y1148) antibody (HA500580) at 1/10,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.