Phospho-EGFR (T669) Recombinant Rabbit Monoclonal Antibody [PSH10-03] - BSA and Azide free

☑ Cell treatment (CT)

Western blot analysis of Phospho-EGFR (T669) on different lysates with Rabbit anti-Phospho-EGFR (T669) antibody (HA751333) at 1/5,000 dilution.

Lane 1: A431 cell lysate
Lane 2: A431 treated with 100ng/mL EGF for 30 minutes cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 134 kDa
Observed band size: 150 kDa

Exposure time: 3 minutes; 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 (HA751333) at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Phospho-EGFR (T669) was immunoprecipitated from 0.2 mg A431 starved overnight then treated with 100ng/mL EGF for 15 minutes cell lysate with HA751333 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA751333 at 1/2,000 dilution. HRP Conjugated Anti-Rabbit IgG for IP Nano-secondary antibody at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: A431 starved overnight then treated with 100ng/mL EGF for 15 minutes cell lysate (input)
Lane 2: HA751333 IP in A431 starved overnight then treated with 100ng/mL EGF for 15 minutes cell lysate
Lane 3: Rabbit IgG instead of HA751333 in A431 starved overnight then treated with 100ng/mL EGF for 15 minutes cell lysate

Blocking/Dilution buffer: primary antibody dilution (K1803)
Exposure time: 59 seconds; ECL: K1801