Phospho-EGFR (Y992) Recombinant Rabbit Monoclonal Antibody [PSH23-63]

☑ Cell treatment (CT)

Western blot analysis of Phospho-EGFR (Y992) on different lysates with Rabbit anti-Phospho-EGFR (Y992) antibody (HA724388) at 1/2,000 dilution.

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

Lysates/proteins at 20 µg/Lane.
Exposure time: 12 seconds; ECL: K1801

Blocking: 5% NFDM/TBST, 1 hour at room temperature
Primary antibody: HA724388, 1/2,000 in primary antibody dilution buffer (K1803), 2 hours at room temperature
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 cells) treated with EGF(100ng/ml 30min)

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: HA724388, 1/1,000, 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)

Application: Flow Cytometry (Intra)

Species: Human
Sample: A431 (Human epidermoid carcinoma skin squamous cells) treated with EGF(100ng/ml 30min)

Fixation: 4% Paraformaldehyde, 15 minutes at room temperature.
Permeabilization: 1x Permeabilization Buffer (eBioscience), 15 minutes at room temperature.

Blocking: 1% BSA + 10% normal goat serum, 15 minutes at room temperature.
Antibody dilution buffer: 1x PBS.
Primary antibody: HA724388 (1/10,000) (Red) compared with Rabbit IgG Isotype Control (HA722127, Green), 15 minutes at room temperature.
Secondary antibody: Goat Anti-Rabbit IgG (iFluor™ 488, HA1121), 15 minutes at room temperature.