HTRF phospho-HER2 (Tyr1221/1222) assay compared to Western Blot

Human SKOV3 cells were grown in a T175 flask at 37°C until 80% confluency. Cells were then stimulated with 100 nM EGF for 10min. After medium removal, the cells were lysed with 3 mL of supplemented lysis buffer for 30 min at room temperature. Soluble supernatants were collected after a 10 minute centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 16 µL of each dilution were dispensed and analyzed side-by-side by Western-blot and by HTRF. By using HTRF phospho-HER2 (Tyr 1221/1222), only 1500 cells are sufficient for minimal signal detection while 12,500 cells are needed for a Western Blot signal. The HTRF assay is at least 8-fold more sensitive than the Western Blot and shows optimal correlation.

 

 

Phospho-HER2 modulation in several human tumor cell lines

100,000 Human SKOV3, SK-BR-3, A431 and MCF-7 cells were plated in 96 well plate and incubated for 24h, at 37°C-5%Co2. After incubation with increasing concentrations of mEGF (10min), medium was removed and cells were lysed with 50µl of Lysis buffer for 30min at RT under gentle shaking. 16 µL of lysate was transfered into a 384-well sv white microplate and 4 µL of the HTRF phospho-HER2 detection reagents were added. HTRF signal was recorded after an Overnight incubation.

 

phospho-HER2 Inhibition by Tyrosine kinase inhibitors and mAbs

Experiments were run on SKOV3 cells. 100,000 cells were plated in 96-well plate and incubated for 24h, at 37°C-5%Co2, then, after a pre-treatment with a dose-response of inhibitors: Trastuzamab, Cetuximab and Pertuzumab (Therapeutic monoclonal antibodies) and Lapatinib (Tyrosine Kinase Inhibitor), the cells were stimulated with mEGF for 10 min at 37 °C, 5% CO2. After these steps, medium was removed and cells were lysed with 50 µL of Lysis buffer for 30min at RT under gentle shaking.16 µL of lysate was transferred into a 384-sv white microplate for detection and 4 µL of the HTRF phospho-HER2 detection reagents were added. HTRF signal was recorded after an Overnight incubation. Results show the applicability of the HTRF phospho-HER2 assay to decipher the mechanism of action of small molecules and biologics targeting HER2.