Phospho-EGFR (Y1110) Recombinant Rabbit Monoclonal Antibody [PSH27-58]

☑ Cell treatment (CT)

Western blot analysis of Phospho-EGFR (Y1110) on different lysates with Rabbit anti-Phospho-EGFR (Y1110) antibody (HA724705) at 1/5,000 dilution.

Lane 1: A431 (Human epidermoid carcinoma skin squamous cell) cell lysate
Lane 2: A431 treated with 100ng/mL EGF for 30 minutes cell lysate

Lysates/proteins at 15 µg/Lane.
Exposure time: 42 seconds; ECL: K1801

Blocking: 5% NFDM/TBST, 1 hour at room temperature
Primary antibody: HA724705, 1/5,000 in primary antibody dilution buffer (K1803), overnight at 4 °C
Secondary antibody: Goat anti-Rabbit IgG-HRP (HA1001), 1/50,000 in 5% NFDM/TBST, 1 hour at room temperature

Predicted band size: 134 kDa
Observed band size: 175 kDa

☑ Cell treatment (CT)

Application: Immunocytochemistry (IF-cell)

Species: Human
Sample: A431 (Human epidermoid carcinoma skin squamous cell) treated with or without 100ng/mL EGF for 30 minutes

Fixation: 4% Paraformaldehyde, 15 minutes at room temperature.
Permeabilization: 0.1% Triton X-100, 15 minutes at room temperature.
Blocking: 1% BSA + 10% normal goat serum, 1 hour at room temperature.
Antibody dilution buffer: 1% BSA in PBST.

Primary antibody: HA724705, 1/500, overnight at 4°C.
Secondary antibody: Goat Anti-Rabbit IgG (iFluor™ 488, HA1121), 45 minutes at room temperature.
Counterstain: Beta tubulin (HA601187, Red), 1/100, overnight at 4℃. The nuclear counterstain was DAPI (Blue).

☑ Cell treatment (CT)

Immunoprecipitation (IP)

Phospho-EGFR (Y1110) was immunoprecipitated in 0.2 mg A431 (Human epidermoid carcinoma skin squamous cell) treated with 100ng/mL EGF for 30 minutes cell lysate with HA724705 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA724705 at 1/5,000 dilution. HRP Conjugated Rabbit IgG kappa light chain Mouse Monoclonal Antibody (M1208-2) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: A431 treated with 100ng/mL EGF for 30 minutes cell lysate (input)
Lane 2: HA724705 IP in A431 treated with 100ng/mL EGF for 30 minutes cell lysate
Lane 3: Rabbit IgG instead of HA724705 in A431 treated with 100ng/mL EGF for 30 minutes cell lysate

Exposure time: 30 seconds
Blocking: 5% NFDM/TBST, 1 hour at room temperature

Primary dilution: HA724705, 1/5,000 in primary antibody dilution buffer (K1803), 2 hours at room temperature

Predicted band size: 134 kDa
Observed band size: 175 kDa