HTRF assay compared to Western Blot using phospho-Smad1 cellular assay on mouse C2C12 cells

Mouse C2C12 cells were cultured to 80% confluency. After BMP-4 treatment, cells were lysed and soluble supernatants were collected via centrifugation. Serial dilutions of the cell lysate were performed and 16 µL of each dilution were transferred into a 384-well low volume white microplate before finally adding phospho kit reagents. A side by side comparison showed the HTRF Phospho assay is at least 8-fold more sensitive than the Western Blot, and shows optimal correlation.

 

 

Validation of the HTRF phospho-SMAD1 (Ser463/465) cellular assay on mouse and human cell lines

Cells were plated in a 96-well plate and incubated for 24h at 37°C, 5% COo2. After treatment for 30 min with BMP-4, cells were lysed, with 50 µL of lysis buffer added for 30min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho-SMAD1 (Ser463/465) detection reagents were added. The HTRF signal was recorded after a 4 hour incubation. Human cervical cancer HeLa cells were plated at 50,000 cells/ well and treated with human BMP-4. Mouse myoblast C2C12 cells were plated at 25,000 cells/ well and treated with mouse BMP-4.